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The Journal of Immunology, 1998, 160: 273-283.
Copyright © 1998 by The American Association of Immunologists

Common and Distinct Signaling Pathways Mediate the Induction of TNF-{alpha} and IL-5 in IgE Plus Antigen-Stimulated Mast Cells

R. Csonga, E. E. Prieschl, D. Jaksche, V. Novotny and T. Baumruker1

Department of Immunology, Novartis Research Institute, Vienna, Austria


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A small number of signaling cascades represented by mitogen-activated protein kinases, phosphoinositol-3-kinase, protein kinase C, signal transducers and activators of transcription, Ca2+/calcineurin, and a few other molecules are linked to an incomparably large number of surface receptors. Parallel activation of several of these pathways and the existence of isozymes for a number of signal transmitting molecules generate the required complexity and specificity matching the receptor variety. Here we show that the proinflammatory mediator TNF-{alpha} and the growth factor IL-5 are activated along common and distinct signaling cascades in allergically stimulated murine mast cells. Both of them are dependent on Ca2+ influx, activation of calcineurin and nuclear factor of activated T cells as well as a member of the atypical PKC family, most likely PKCµ. Additionally, mitogen-activated protein kinases for TNF-{alpha} and members of the classical or nonclassical PKCs for IL-5, respectively, were identified as additional required pathways. Inhibition of the classical and nonclassical PKCs, however, does not abrogate IL-5 induction but instead leads to a switch to mitogen-activated protein kinases, which then become essential. The activated branches of this "salvage" signaling cascade are represented by extracellular signal-regulated kinase 1/2 and c-jun NH2 terminal kinase 1 in allergically stimulated mast cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Fc{epsilon}RI proximal signaling events are well established in mast cells after IgE plus Ag triggering. Two protein tyrosine kinases, lyn and syk, interact with the ß- and {gamma}-chain of the tetrameric receptor complex, respectively, and initiate a first wave of intra- and intermolecular tyrosine phosphorylation (1, 2, 3). Other tyrosine kinases such as Btk, Emt, c-src and c-yes are thought to further amplify and transfer this initial signal to the cytoplasmic components of the various signaling cascades (4, 5). Several lines of evidence suggest that one of these activated pathways uses the mitogen-activated protein kinases (MAPKs)2 (6, 7, 8) that comprise a complex signaling system, which in large part is evolutionarily highly conserved between yeast and mammals (9). Currently, three branches of this system are known, with erk (extracellular signal-regulated kinase), jnk (c-jun NH2 terminal kinase), and p38 kinase being the end points of this cascade (10). They exist in different isoforms and splice variants (11). In contrast to yeast, in which each branch regulates separate phenomena and no parallel activation by the same stimulus occurs, in mammals simultaneous activation of several branches by one stimulus is frequently observed (9). Engagement not only of growth factor receptors but also of the TCR, the B cell receptor (BCR), and the Fc{epsilon}RI in mast cells provides the initial trigger (7, 12, 13). Induction, phosphorylation/dephosphorylation, and nuclear import of transcription factors such as ATF2, elk1, jun, and SRF comprise the final steps in these cascades for gene activation (14).

In mast cells lymphokines and chemokines are induced under the control of NF-AT transcription factor family members (15) in conjunction with different inducibly and constitutively expressed cofactors (16, 17). The FK506 sensitivity of this process suggests that, as in T cells, NF-AT is activated along the Ca2+ influx/calcineurin pathway in mast cells. The additional contribution of the MAPK pathways, especially regarding the cofactors, with respect to induction, phosphorylation/dephosphorylation, and nuclear import, is not fully clarified so far.

Here, we show that the transcription of TNF-{alpha} is controlled by an NF-AT transcription factor family member plus an activator protein (AP)1-like cofactor after the IgE plus Ag trigger. Transdominant negative mutants of p21ras (ras N17) as well as the MAPK pathway inhibitors Apigenin and PD98059 specifically block the induction of TNF-{alpha}. IL-5, which is independent of the AP1-like cofactors for its activation, is not affected by inhibition of the MAPK pathway by Apigenin or the transdominant negative ras mutant (16). Instead, IL-5 is induced by PMA-dependent PKCs and, only alternatively, in the case of their inhibition, are MAPK pathways used for the activation. Both TNF-{alpha} and IL-5 need for their activation a pathway that is sensitive to Gö 6976, whose most likely target is the atypical (PMA- and Ca2+-independent) PKC member µ (18).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culturing of CPII mouse mast cells, generation of radiolabeled probes, electrophoretic mobility shift assays, murine TNF-{alpha} ELISAs, and transient transfections of DC18 cells were done as recently described (17, 19, 20, 21).

Transient transfection of CPII mouse mast cells

Cells were treated with 0.25% trypsin for 5 min prior to electroporation. Ten micrograms of reporter gene construct together with 2 µg of pRL-TK (thymidine kinase promoter-dependent renilla luciferase construct; Promega, Madison, WI) were used for determination of transfection efficiency and cell recovery. In the case of cotransfections, 6 µg of reporter gene construct, 10 µg of the cotransfected plasmid (ras N17 or v-ras), and 2 µg of pRL-TK were transfected together. Transfections were done using 8 x 106 CPII cells in 250 µl medium in a 0.4-cm gap electroporation cuvette (Bio-Rad Laboratories, Hercules, CA). Electroporation was carried out at 230 V, 960 µF, with an average time constant of 55. Ten microliters of the transfected cells were plated in a 48-well plate (Costar, Cambridge, MA) and 500 µl fresh medium was added to each well. Thirty-six hours after transfection, cells were stimulated as indicated by either 2 µg/ml monoclonal mouse IgE anti-TNP/DNP (PharMingen, San Diego, CA) and 100 ng/ml Ag (DNP-BSA) (Calbiochem Corp., La Jolla, CA) or with 20 ng/ml PMA (Sigma Chemical Co., St. Louis, MO) and/or 100 ng/ml ionomycin (Sigma). From 12 to 15 h later a dual luciferase assay (Promega) was performed as described by the manufacturer.

Western blot analysis

Cells were lysed in sample buffer (Novex, San Diego, CA) containing 2% ß-mercaptoethanol. Twenty microliters were loaded on precast Tris-Glycine 4 to 20% gradient gels (Novex). Proteins were transferred to polyvinylidene difluoride membranes (Novex) by electroblotting. Membranes were blocked with 5% skim milk in PBS containing 0.1% Tween-20 (Bio-Rad) and incubated with the primary Ab at 4°C overnight. The Abs for erk 1/2, p-erk 1/2, jnk1/2, p-jnk1/2, MEK1/2, p-MEK1/2, p38, and p-p38 were purchased from New England Biolabs, Beverly, MA); anti-phosphothreonine was purchased from Sigma. After being washed three times with PBS-Tween, membranes were incubated at room temperature for 1 h with the second Ab (anti-rabbit IgG/alkaline phosphatase for erk1/2, p-erk1/2, jnk1/2, p-jnk1/2, MEK1/2, p-MEK1/2, p38, and p-p38 (New England Biolabs) and goat anti-mouse horseradish peroxidase conjugated for PKCµ and anti-phosphothreonine (Bio-Rad) and washed four times with PBS-Tween. Detection by chemiluminescence (CDP Star for erk1/2, p-erk1/2, jnk1/2, p-jnk1/2, MEK1/2, p-MEK1/2, p38, and p-p38 (New England Biolabs), and ECL for PKCµ and anti-phosphothreonine (Amersham, Little Chalfont, UK)) was performed as described by the manufacturer.

Immunoprecipitation and in-gel kinase assay

A total of 107 CPII cells were lysed in 0.5 ml extraction buffer (1% SDS, 1 mM Na3VO4, and 10 mM Tris/HCl, pH 7.4) by boiling for 5 min and squeezing three times through a 26-gauge needle. The cleared supernatant of this denatured total cell lysate was diluted 10-fold with immunoprecipitation buffer (1% Triton X-100, 0.5% NP-40, 10 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2 mM Na3VO4, and 0.2 mM PMSF). One milliliter of the diluted lysate was incubated with 2 µg anti-PKCµ Ab (Transduction Laboratories, Lexington, KY) for 1 h at 4°C. Five micrograms of rabbit anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) were added and incubated for a further 30 min. Finally, 10 µl protein A-agarose beads (Transduction Laboratories) was added and incubated with agitation for another 30 min. Agarose beads were washed twice with immunoprecipitation buffer and resuspended in 2 x SDS PAGE sample buffer (Novex), boiled for 5 min and centrifuged. The supernatants were subjected to a gel electrophoresis on precast Novex gels as described before. SDS was removed by washing the gel three times for 20 min in 50 mM Tris/HCl, pH 8, and 20% isopropanol, and once for 60 min in 50 mM Tris/HCl, pH 8, and 5 mM DTT. The gel was denatured with 6 M guanidine HCl, 50 mM Tris/HCl, pH 8, and 5 mM DTT. Renaturation was done overnight with 50 mM Tris/HCl, pH 8, 5 mM DTT, and 0.04% Tween-20. The gel was preincubated in 50 mM Tris/HCl, pH 8, 5 mM DTT, 4 mM MgCl2, and 100 µg/ml phosphatidylserine for 20 min before adding 2 µCi/ml [{gamma}-32P]ATP (Amersham) with or without the inhibitor. After 1 h the kinase reaction was stopped by washing the gel in 5% TCA, 1% sodium pyrophosphate. The gel was dried and subjected to autoradiography.

Inhibitors

Cells were incubated with Apigenin (Calbiochem), PD98059 (Calbiochem), or Gö 6976 (Calbiochem) for 1 h prior to stimulation at the concentrations indicated. All substances were dissolved in DMSO. Solvent control is DMSO at a concentration equivalent to the lowest dilution of the drug.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mapping of the TNF-{alpha} promoter after an IgE plus Ag stimulus

Recently we showed that TNF-{alpha} is transcriptionally induced in the mast cell line CPII after the artificial stimulus PMA plus ionomycin. NF-AT and an AP1-like cofactor bind to and control the {kappa}3 site of its promoter (17). We started out to readdress the question of TNF-{alpha} induction, this time after the more physiologic stimulus of IgE plus Ag. By using the same 5' successive deletions as in our previous study, the responsible region of the promoter was again mapped to the 5' extended {kappa}3 site (Fig. 1Go, A and B). Partial deletion of the AP1 site adjacent to the {kappa}3 site—as in deletion 2, where the first T of the AP1 site is missing—completely abolished functionality. Consistent with this result and as also seen after PMA plus ionomycin stimulation, we identified an NF-AT family member (competition with an NF-AT consensus site from the murine IL-2 promoter) and an AP1-like factor (competition with an AP1 consensus site and an NF-E2 consensus site containing an AP1-binding sequence) in a competition analysis as inducible factors binding to this 23-bp long probe (Fig. 1GoC). From these data we conclude that the involved regulatory sequences and the participating transcription factors are indistinguishable after PMA plus ionomycin compared with IgE plus Ag stimulation.



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  		FIGURE 1. Mapping of the TNF-{alpha} promoter after IgE plus Ag stimulation. A, A map of the TNF-{alpha} promoter linked to luciferase is shown. The relative distance in bp to the start site of transcription is given above, consensus binding sites for transcription factors are indicated underneath. The three 5' successive deletions used in the mapping are schematically shown. B, Transient transfection with the deletions. Uneven lanes represent noninduced, even lanes induced, values. Luciferase values are indicated at the y-axis; deletions are given at the x-axis. All experiments were performed in triplicate; SD is indicated. C, Gel shift analysis with the {kappa}3 site. Noninduced indicates nuclear extracts of nonstimulated, induced nuclear extract of IgE plus Ag-stimulated CPII cells. Competitor consensus-binding sites in x-fold excess are indicated above the gel shift. NF-E2 is a consensus-binding site for nuclear factor-erythroid containing an AP1-binding sequence.

 
Activation of the MAPK pathway after IgE plus Ag stimulation in mast cells

The MAPK pathways comprise alternative, parallel (somehow interrelated) signaling cascades to the PKC pathway and are known to activate a number of transcription factors capable of binding to AP1-sites, like fos and jun (22, 23). Therefore, and based on recent experiments suggesting a direct linkage of the Fc{epsilon}RI to p21ras and the raf kinase (6, 7), we made an inventory of the three MAPK pathways in allergically activated mast cells. By Western blot analyses the expression and phosphorylation of erk 1/2 (Fig. 2GoA), jnk 1/2 (Fig. 2GoB), and p38 (Fig. 2GoC) were determined after various time points of induction. It is clearly visible that erk 1/2 are constitutively present in our CPII cell line and become rapidly phosphorylated 5 min after stimulation. Peak values of phosphorylation (activation) are seen at around 15 min, with the activation status declining afterward (Fig. 2GoA, right panel). Jnk 1/2 are also expressed constantly with jnk 1 becoming phosphorylated at around 10 min of activation and declining in its phosphorylation after 15 min. No activation of jnk 2 was detected (Fig. 2GoB, right panel). p38 MAPK is constitutively found in CPII mast cells, but in contrast to erk 1/2 and jnk 1 its phosphorylation is, if at all, only slightly enhanced after the allergic trigger (Fig. 2GoC, right panel; see Discussion). From these data we conclude that erk 1/2 and jnk 1 are the activated MAPKs in CPII cells after IgE plus Ag stimulation.



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  		FIGURE 2. The MAPK pathway activation in IgE plus Ag-stimulated CPII cells. A, Western blot analysis over activation time for erk 1/2 expression/activation. Left, Constitutive erk 1/2 protein expression; right, phospho-erk 1/2 protein expression. The time points of stimulation before preparing the cellular extracts are indicated above the lanes. B, Same experimental setting as under A for the detection of jnk 1/2 (left) and phospho-jnk 1/2 (right). C, Same experimental setting as under A for the detection of p38 (left) and phospho-p38 (right).

 
Apigenin comprises a specific inhibitor at the level of MAPKs

Several low m.w. inhibitors of the MAPK pathway were recently characterized. They enable investigations at functional and biochemical levels simultaneously. Apigenin, a flavoid, is able to reverse the growth of ras-transformed cells with an IC50 of 25 µM and is considered to be a specific ras/MAPK pathway inhibitor at the level of MAPKs (24, 25). Application of Apigenin at 30 µM, 1 h prior to IgE plus Ag stimulation, prevented the phosphorylation of erk 1/2 (Fig. 3GoA, right panel) and jnk 1 (Fig. 3GoB). In contrast, no effect of Apigenin at this concentration was detected concerning the phosphorylation of MEK 1/2 (MAPK kinase), which comprises one level of regulation above erk 1/2 (Fig. 3GoC). Therefore we concluded that Apigenin at around 30 µM disrupts the signal transduction of the MAPK pathways relatively specifically (for functional specificity see later). It comprises a useful tool to investigate the contribution of this signaling cascade to the overall activation of cytokines.



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  		FIGURE 3. Inhibition of erk 1/2 and jnk 1 activation by Apigenin. A, Effects of Apigenin on erk 1/2 activation. Left, constitutive erk 1/2 expression; right, phospho-erk 1/2 expression. Extracts of cells nonstimulated (nonstimulated), stimulated for 15 min with IgE plus Ag (stimulated), or stimulated for 15 min with IgE plus Ag and pretreated 1 h before with Apigenin (30 µM Apigenin) or solvent (solvent control) are shown. B, Same experimental setting as under A for the detection of phospho-jnk 1. C, Same experimental setting as under A for the detection of phospho-MEK 1/2 as a specificity control.

 
Inhibition of the MAPK pathway blocks TNF-{alpha} gene induction

To establish a first link of the MAPK pathways to the overall transcriptional induction of TNF-{alpha}, we performed transient transfections with our reporter gene construct together with a transdominant negative mutant of ras (ras N17), as well as a constitutively active form (v-ras) (Fig. 4GoA). The active form of ras strongly synergized with the Ca2+ ionophore ionomycin resulting in induction levels equal to the IgE plus Ag stimulus. Neither of them alone, however, was able to activate the TNF-{alpha} reporter gene. This is coherent with a picture in which NF-AT is exclusively activated along the Ca2+ pathway, while the required cofactors would be (only) induced by the ras/MAPK pathway. Cotransfection of the transdominant negative ras mutant leads to a more than 50% reduction of the stimulation of the TNF-{alpha} reporter gene by IgE plus Ag. These results together imply that activation of the MAPK pathway is required for the transcriptional induction of TNF-{alpha}.



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  		FIGURE 4. Effects of MAPK pathway blockage on TNF-{alpha} gene induction. A, Cotransfection with a constitutively active (v-ras) and transdominant negative (ras N17) ras mutant. Luciferase values are shown on the y-axis; stimuli and input plasmid DNA are given at the x-axis. pC-RSV and pGL2 are the parental vectors. TNF-luciferase-TNF-3' is the reporter gene construct. All experiments were done in triplicate; SD is indicated. B, Transient transfection with and without Apigenin application 1 h prior to stimulation. Luciferase values are shown on the y-axis; stimuli, inhibitors, and input plasmid DNA are given at the x-axis. pGL2 is the parental vector, TNF-luciferase-TNF-3' is the reporter gene construct. All experiments were done in triplicate; SD is indicated.

 
To further clarify whether ras mediates TNF-{alpha} activation via the MAPK cascade or by different mechanisms (based on its pleiotropic actions) (10) we applied Apigenin at 10 µM and 30 µM concentrations in transient transfection assays 1 h prior to IgE plus Ag stimulation. The drug dose dependently inhibited the transcriptional induction of TNF-{alpha} (Fig. 4GoB), therefore linking the MAPK pathways to the induction of TNF-{alpha} in allergically activated mast cells.

Erk 1/2 inhibition prevents TNF-{alpha} induction

Compound PD 98059, also a flavone-type substance as Apigenin, is known as a specific erk 1/2 inhibitor. Recently, Zhang et al. (26) found that it prevents TNF-{alpha} production in RBL-2H3 cells with an IC50 of around 20 µM, while Ishizuka et al. (27) detected no effect up to 30 µM of this drug using the same readout in MC/9 cells. Transcriptional activation of TNF-{alpha}, as measured in the reporter gene assay in CPII cells was strongly inhibited by PD 98059 (IC50 = 2.5 µM) at concentrations similar to the described IC50 values for erk 1/2 inhibition (Fig. 5GoA) (28). TNF-{alpha} production, as measured in an ELISA, was also effected, however, at 10 times higher concentrations, which is in the same range as Zhang et al. reported for RBL cells (Fig. 5GoB) (26). The effect of PD 98059 supports a functional involvement of erk 1/2 in the signaling process in CPII cells, with the difference in IC50 values for transcriptional and secretional inhibition most likely being due to the preformed TNF-{alpha} in mast cells (see Discussion).



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  		FIGURE 5. Inhibition of TNF-{alpha} production by PD 98059. A, Transient transfection assay with a TNF-{alpha}-luciferase-TNF-{alpha}-3' reporter gene construct. Luciferase values are shown on the y-axis; stimuli, inhibitor, and concentration of inhibitor are given at the x-axis. All experiments were done in triplicate; SD is indicated. B, TNF-{alpha} ELISA with and without Apigenin and PD 98059 application 1 h prior to stimulation. Production of TNF-{alpha} in nanograms per milliliter is indicated at the y-axis; stimuli, inhibitors, and concentration of inhibitors are given at the x-axis. All experiments were done in triplicate; SD is indicated.

 
TNF-{alpha} but not IL-5 induction is affected by MAPK pathway blockage

To elucidate which component of the transcription factor complex is affected by disrupting the MAPK pathway, we performed an identical set of experiments using a reporter gene construct driven by a minimal promoter under the exclusive control of three AP1 sites (3 x TRE) (17). Its induction is inhibited to the same degree as observed for the TNF-{alpha} promoter construct by either cotransfection with the transdominant negative mutant of ras or by Apigenin treatment at identical concentrations (Fig. 6Go, A and B). The activated mutant of ras already led to a full stimulation in nontreated cells and together with IgE plus Ag resulted in an even more enhanced induction than that seen under physiologic conditions. This indicates that the AP1 factor is under the exclusive control of the ras-MAPK cascade in mast cells. It led us to hypothesize that the activation of the inducible cofactors is one of the targets of the MAPK pathway blockage. To prove this further and rule out effects on the NF-AT component, we investigated the IL-5 promoter-mediated induction.



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  		FIGURE 6. Effects of MAPK pathway blockage on AP1. Effects of MAPK pathway blockage on AP1-mediated gene induction. A, Cotransfection with a constitutively active (v-ras) and transdominant negative (ras N17) ras mutant. Luciferase values are shown on the y-axis; stimuli and input plasmid DNA are given at the x-axis. pC-RSV and pGL2 are the parental vectors. 3 x TRE is the reporter gene construct. All experiments were done in triplicate; SD is indicated. B, Transient transfection with and without Apigenin application 1 h prior to stimulation. Luciferase values are shown on the y-axis; stimuli, inhibitors, and input plasmid DNA are given at the x-axis. pGL2 is the parental vector, 3 x TRE is the reporter gene construct. All experiments were done in triplicate; SD is indicated.

 
In CPII mast cells, the activation of this promoter was recently characterized to be dependent on an NF-AT family member, in co-operation with a constitutively expressed GATA family member after IgE plus Ag stimulation (16). Therefore, it should be insensitive to the blockage of the MAPK pathway, if the hypothesis is correct. A cotransfection with the transdominant negative mutant of ras and the IL-5 reporter gene plasmid resulted in no statistically significant inhibitory effect, in contrast to the findings with the TNF-{alpha} and the AP1-driven constructs (Fig. 7GoA). Also, no inhibition was observed upon the application of the MAPK pathway inhibitor Apigenin, which at the lower concentration even results reproducibly in an enhancement of induction (Fig. 7GoB). It is, however, noteworthy that the activated ras mutant induces IL-5 transcription in noninduced cells to a significant degree and again strongly synergizes with ionomycin in the induction. This implies that ras-dependent signaling cascades can be an alternative, but usually not essential, pathway in the full activation of this cytokine (see later).



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  		FIGURE 7. Effects of MAPK pathway blockage on IL-5. Effects of MAPK pathway blockage on IL-5 gene induction. A, Cotransfection with a constitutively active (v-ras) and transdominant negative (ras N17) ras mutant. Luciferase values are shown on the y-axis; stimuli and input plasmid DNA are given at the x-axis. pC-RSV and pGL2 are the parental vectors. IL-5 is the reporter gene construct. All experiments were done in triplicate; SD is indicated. B, Transient transfection with and without Apigenin application 1 h prior to stimulation. Luciferase values are shown on the y-axis; stimuli, inhibitors, and input plasmid DNA are given at the x-axis. pGL2 is the parental vector; IL-5 is the reporter gene construct. All experiments were done in triplicate; SD is indicated.

 
An atypical PKC member is needed for both TNF-{alpha} and IL-5 gene activation

We were next interested in characterizing (essential) signaling cascades involved in IL-5 induction to further differentiate the activation of this growth factor from the activation of the proinflammatory TNF-{alpha} mediator. As we have recently shown, TNF-{alpha} and IL-5 are both sensitive to FK506 and are therefore activated along the Ca2+/calcineurin/NF-AT pathway (16, 17). The finding that ionomycin clearly, but to a lesser extent than IgE plus Ag, activates IL-5 suggested that at least a second pathway is required for full activation as also shown for TNF-{alpha}. The synergy of active ras with ionomycin suggested that MAPKs have the potential to contribute to IL-5 regulation. In contrast, IL-5 activation is not affected by cotransfection of a transdominant negative mutant of ras and Apigenin application, implying that the MAPK pathways are not necessarily required for its induction. An alternative route is comprised of PKCs. PMA-dependent PKCs (classical and nonclassical ones) can be eliminated in mast cells by depleting these kinases via a prolonged (48-h) pretreatment with PMA (6). Transient transfection assays with nondepleted and PKC-depleted mast cells showed that neither TNF-{alpha} nor IL-5 induction depends on classical or nonclassical PKCs after IgE plus Ag stimulation, while both mediators in the control experiment were no longer activated in response to PMA plus ionomycin in the depleted cells (Fig. 8GoA). Surprisingly, Apigenin, which had no effect on IL-5 induction in normal CPII cells, blocks its up-regulation in PKC-depleted cells to basal levels of expression (Fig. 8GoB). This indicates that in contrast to TNF-{alpha}, IL-5 can be induced either by PMA-dependent PKCs or by the MAPK pathway as an alternative route (see Discussion).



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  		FIGURE 8. Classical and nonclassical PKCs are involved in IL-5 activation. A, Transient transfections of nondepleted and PKC-depleted CPII cells. Plasmid DNA for transfection and stimuli is shown on the x-axis; luciferase values are given at the y-axis. All experiments were done in triplicate; SD is indicated. The cell source—nondepleted or depleted CPII cells—is indicated above the panels. B, Use of Apigenin in transient transfections of nondepleted and depleted CPII cells. Plasmid DNA for transfection, stimuli, and inhibitors is shown on the x-axis; luciferase values are given at the y-axis. All experiments were done in triplicate; SD is indicated. The cell source—nondepleted or depleted—is indicated above the panels.

 
To further differentiate the PMA-dependent PKCs into the classical and the nonclassical subfamily, we applied Gö 6976. This compound is characterized to be a specific inhibitor of the classical PKCs and, in addition, was recently shown to inhibit also the atypical PKC member µ at a concentration of 2 µM (18, 29). Untreated CPII cells as well as PKC-depleted ones are affected to the same degree by this inhibitor and no difference was found for the two cytokines under investigation, TNF-{alpha} and IL-5 (Fig. 9Go). This indicates that, in addition to the MAPK pathway for TNF-{alpha} and the PMA-dependent PKC pathway for IL-5, a Gö 6976 sensitive step/pathway, most likely via PKCµ, might be involved in the induction of both mediators. To verify the action of Gö 6976 on PKCµ in our mast cells, this kinase was immunoprecipitated from CPII cell extracts and its autophosphorylation in vitro and its threonine and serine phosphorylation status in vivo with and without the drug were investigated. At 2 µM concentrations, this inhibitor strongly prevented autophosphorylation of immunoprecipitated PKCµ in an in-gel kinase assay when applied during the in vitro reaction (Fig. 10GoA). No difference, however, was seen between drug pretreated and nontreated cells (data not shown). This is in agreement with the chemical nature of the drug and a reversible mode of inhibition.



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  		FIGURE 9. Gö 6976 defines a common signaling pathway/molecule. Use of Gö 6976 in transient transfections of nondepleted and PKC-depleted CPII cells. Plasmid DNA for transfection, stimuli, and inhibitors is shown on the x-axis; luciferase values are given at the y-axis. All experiments were done in triplicate; SD is indicated. The cell source—nondepleted or depleted—is indicated above the panels.

 


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  		FIGURE 10. Gö 6976 prevents PKCµ activity and phosphorylation. A, In-gel kinase assay either in the absence (nontreated) or presence of 2 µM Gö 6976 (2 µM Gö 6976). Activity by measuring autophosphorylation of immunoprecipitated PKCµ (top band) of nonstimulated and 30-min stimulated CPII cells is shown. B, Western blot analysis of immunoprecipitated PKCµ from CPII cells. Left, Western blot with anti-PKCµ Ab as loading/precipitation control. Right, Western blot with anti-phosphothreonine Ab. Stimulation and use of inhibitor are indicated above the panels. PKCµ and Abs used in the immunoprecipitation are indicated to the right. Std. is a size standard. C, Gö 6976 has no effect on activated (IgG/Ag) DC18 APCs. A Transient transfection assay of DC18 cells with the TNF-{alpha}-luciferase-TNF-{alpha}-3' reporter gene construct is shown. Luciferase values are given at the y-axis; stimuli, inhibitor, and concentration of inhibitor are given at the x-axis. All experiments were done in triplicate; SD is indicated.

 
To prove the action of Gö 6976 in vivo (in cells), we analyzed the phosphorylation status of PKCµ in nonstimulated and stimulated cells with and without drug treatment. While IgE plus Ag triggering induced a threonine and serine phosphorylation (the latter very weak, data not shown) of PKCµ 30 min after stimulation, this modification was strongly inhibited in Gö 6976 pretreated cells (Fig. 10GoB). It proves that at a concentration of 2 µM, murine mast cell-derived PKCµ is a target of this compound (see Discussion). TNF-{alpha} transcriptional activation of the reporter gene construct in an Ag-presenting mouse dendritic cell line (DC18) was not inhibited after triggering, indicating the specificity of the reaction (Fig. 10GoC).

Gö 6976, but not Apigenin disrupts the complex formation at the TNF-{alpha} promoter

While these data can be interpreted as showing that the two inhibitors (Apigenin and Gö 6976) define two different signaling pathways after IgE plus Ag stimulation, alternative explanations can also be drawn. The strong inhibition of the AP1-driven reporter gene construct by Gö 6976 (Fig. 11GoA), as well as the finding that it completely prevents the phosphorylation of erk 1/2 (Fig. 11GoB), would also be in agreement with a mode of action of this compound similar to Apigenin and additional inhibition of the classical and nonclassical PKCs. In this case, TNF-{alpha} and IL-5 would both be inhibited. Recent findings in other cell types have proved that erk 1/2 is involved in the activation of components of AP1 complexes (14). This step is not necessary for the nuclear import or the binding to the promoter sites of the AP1 factor. Therefore, AP1-like cofactors might still be found in the nucleus in an indistinguishable composition, binding to the {kappa}3 site, after stimulation, if only the MAPK pathway is inhibited. If Gö 6976 defines an additional activation pathway required for a different step of transcription factor activation, the picture in contrast should be different compared with Apigenin treatment. Nuclear extracts with and without application of both drugs were used in a gel shift analysis with the {kappa}3 site as radiolabeled probe. This ruled out effects of the drug mediated by its inhibition of classical and nonclassical PKCs, of which TNF-{alpha} is independent. Clearly visible is the fact that the pattern of complex formation under Apigenin application is indistinguishable from that of nontreated or solvent-treated cells while Gö 6976 application results in a different pattern of complex formation after stimulation. Here, not only the faster migrating NF-AT complex is strongly diminished, but more prominently, the two AP1-like complexes have disappeared and a new complex with different mobility is found (Fig. 11GoC). This clearly differentiates the inhibition by Gö 6976 from a pure MAPK mode of action.



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  		FIGURE 11. Apigenin and Gö 6976 define different interaction points in the signaling cascade. A, Transient transfection with an AP1 reporter gene plasmid with and without Gö 6976 treatment. Stimuli and inhibitors are shown at the x-axis; luciferase values are given at the y-axis. All experiments were done in triplicate; SD is indicated. B, Western blot analyses of phospho-erk1/2 after Apigenin and Gö 6976 treatment. Extracts of nonstimulated, 1 h before stimulation (IgE plus Ag) with Apigenin (30 µM) and Gö 6976 (2 µM) pretreated and solvent-treated cells are shown. C, Gel shift analysis with the two inhibitors, Apigenin and Gö 6976. The source of nuclear extracts of IgE plus Ag-stimulated (induced), nonstimulated (noninduced) CPII cells, and the used inhibitors/solvent control is shown above the lanes. Nuclear extracts were prepared 2 h after stimulation with 1 h pretreatment with the drugs or solvent control. The AP1-like double complex is shown enlarged to the right.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
MAPK, pp90rsk, and pp70-S6 kinase activation were reported in various mast cell systems after Fc{epsilon}RI triggering (7, 26, 27, 30). In T and B cells, the coordinate simultaneous activation of several of the MAPK pathway branches is necessary for a successful response, if triggered by their Ag-recognizing receptor structures (TCR, BCR). Ag encounter of the BCR/TCR on naive B cells/T cells may result in activation, anergy, and apoptosis, depending on the costimulatory signals and therefore on the signaling cascades linked to those structures. If WEHI-231 B lymphoma cells activate only erk 1/2 after BCR stimulation, they undergo apoptosis. Costimulation by CD40/CD40 ligand interaction strongly activates jnk 1 in addition and programs the cells for survival (31). A similar situation holds true for the TCR stimulation and the costimulatory molecule CD28 (32). Even though an anergic or apoptotic pathway after Fc{epsilon}RI triggering in mast cells was never described (with the exception of growth factor deprivation), the structural similarity of the TCR/BCR to the Fc{epsilon}RI made it very likely that several MAPK pathway branches are also activated in mast cells. The recent finding that mast cells express and functionally respond to CD28 triggering—the essential costimulus for T cells linked to jnk activation—raises the question of whether stimulation of the Fc{epsilon}RI alone already confers activation of multiple MAPK pathway branches (33). In contrast to BCR and TCR triggering, Fc{epsilon}RI engagement is sufficient for a profound dual activation of erk 1/2 and jnk 1 in CPII cells. This suggests that the Fc{epsilon}RI on mast cells has a much more dominant role in activation than the related structures on T and B cells and that costimulatory molecules like CD28 (for T cells) or CD40 (for B cells) play only an accessory or amplifying part in mast cells.

The involvement of the MAPK pathways in cytokine gene regulation in B, T, and mast cells was investigated primarily by using transdominant negative mutants of ras and reporter gene technology and only lately by several further downstream acting inhibitors (6, 26, 27, 34, 35). Numerous findings in various cell systems, however, have demonstrated the pleiotropic potential of p21ras, resulting in the activation of signaling molecules outside the MAPK pathways (10). For example, Rac-1 activation via p21ras was recently postulated to participate in the NF-AT activation in this cell type independent of the raf/MEK axis (36). The use of specific low m.w. inhibitors such as Apigenin and PD 98059 further downstream in the MAPK pathway cascade at the present time enables circumvention of this disadvantage. In RBL-2H3 cells and MC/9 cells, however, their recent application has resulted in a noncoherent picture on the details of mast cell activation (26, 27). While in both investigated systems the MAPK pathway was found to be important, erk 1/2 in RBL cells and jnk in MC/9 cells were shown to be involved. The use of Apigenin, which specifically blocks erk 1/2 and jnk 1 activation and the subsequent induction of TNF-{alpha}- and AP1/AP1-like-driven reporter gene constructs in CPII cells, proves a similar linkage of the MAPK pathway to proinflammatory mediator production in our mast cells. The effect of PD 98059, in addition, points toward erk 1/2 participation and therefore makes CPII cells similar to RBL cells. The difference to MC/9 mast cells is, however, not only restricted to this fact. PI3-kinase, which Gelfand and his group (27) found to be important for the activation, was recently shown to play no role in the TNF-{alpha} induction in CPII cells (21). Also, the ineffectiveness of SB 203580, a specific inhibitor for p38 at concentrations up to 30 µM, which is 1000-fold higher than its IC50 is identical to what is found in RBL cells (R. Csonga, unpublished observations). This provides strong evidence that the slight activation of p38, as measured by its phosphorylation after the IgE plus Ag trigger, is not relevant for induction of TNF-{alpha} in the CPII mast cells and might—as Zhang et al. speculated—comprise a negative feedback signal (27).

While our biochemical data with Apigenin support the published claim of specificity of this drug (24, 25), its ineffectiveness in inhibiting IL-5 induction strongly underlines the specific action of this compound. In addition, it characterizes the signaling pathways necessary for activation of this growth factor as being different from those for the proinflammatory cytokine TNF-{alpha}. TNF-{alpha} requires the inducible AP1-like component plus an NF-AT family member while IL-5 uses a constitutively expressed GATA family member as the cofactor for induction. The inhibition of the AP1/AP1-like-driven reporter gene construct by Apigenin implies that the different usage of cofactors comprises the basis for this different requirement in the signaling pathways. This is in agreement with the common hypothesis that NF-AT is induced along the Ca2+/calcineurin pathway and the AP1/AP1-like cofactors along the MAPK pathways. Two lines of evidence, however, suggest that the picture is more complex. First, the strong synergy of ionomycin with the activating mutant of ras in IL-5 activation, which indicates that this additional signaling pathway (also not absolutely required), can contribute to the overall induction of an NF-AT plus GATA-driven promoter. Second, the results of the PMA depletion experiment and the inhibition with Gö 6976 provide evidence for a signal cascade switch and further pathways/molecules being involved.

PKC regulatory enzymes have long been postulated to participate in certain mast cell functions after Fc{epsilon}RI stimulation. Work by Ozawa et al. indicated that PKCß or {delta} but not PKC{alpha} or {epsilon} mediate secretory responses (37). Lewin et al. showed that PKCß and {epsilon}, activated by aggregation of the Fc{epsilon}RI, result in induction of c-fos and c-jun mRNA synthesis. FIP synthesis and DNA-binding activity are stimulated by PKCß (38). All these signaling molecules belong to the PMA-dependent isoforms. Our recent finding that after IgE plus Ag stimulation they are dispensable for the induction of the chemokine MARC seemed to contradict an important function of those kinases in mast cell activation (6). This is even further strengthened by the finding that it also holds true for the two mediators, TNF-{alpha} and IL-5. Variations in the cell lines/primary cells used could be an obvious explanation. The sensitivity of IL-5 to Apigenin in PKC-depleted cells, however, shows that under normal instances PMA-dependent PKCs are/can be involved at least in the induction of certain mediators. IL-5 obviously can be activated via the PKC pathway or alternatively via the MAPK pathway. While our experiments do not clarify to which extent both cascades contribute under normal circumstances they at least prove that they can fully substitute for each other in the induction of this growth factor. This is not the case for TNF-{alpha}, for a currently unknown reason. The identical transcription factors, found after IgE plus Ag or PMA plus ionomycin stimulation, at the {kappa}3 site of the TNF-{alpha} promoter show, however, that PMA-dependent PKC stimulation results in the activation of this proinflammatory mediator in an identical fashion.

The finding that in depleted cells Gö 6976, an inhibitor of the classical PKCs and PKCµ, is able to prevent mast cell cytokine induction implies that a further signaling cascade/signaling molecule is necessary. The threonine and serine phosphorylation of PKCµ after IgE plus Ag stimulation strongly underlines the participation of this kinase in the activation process in our mast cells. It is clearly one of the targets of the inhibitor Gö 6976 at the concentration used, even if we cannot totally rule out that other known or unknown molecules might also be affected. The completely different pattern of complex formation in nuclear extracts of Gö 6976-treated cells, as observed in gel shift analyses with the {kappa}3 site as a radiolabeled probe, clearly distinguishes its inhibitory potential from a pure MAPK inhibition as seen by Apigenin treatment. The appearance of an induced complex in Gö 6976-treated cells after IgE plus Ag stimulation rules out general inhibition (toxicity) by the compound, which is also underlined by noneffected values of renilla luciferase in control plasmids constitutively expressed under the regulation of the tk promoter. In addition, no effect of Gö 6976 (up to 10 µM) is seen on the transcriptional induction of TNF-{alpha} in IgG plus Ag-stimulated DC18 cells. Therefore, molecules targeted by this drug, one of which is PKCµ, comprise a separate activation pathway/signaling molecule in this mast cell line. It is noteworthy that recently PKCµ was found to associate with the BCR complex (39). Here it is up-regulated after cross-linking of the BCR and CD19 and coprecipitates syk and PLC{gamma} 1/2, two important regulators also in mast cells. It is partially regulated in this cell type by Btk, whose activation and phosphorylation in mast cells after Fc{epsilon}RI triggering is documented (5). Atypical PKCs are the target of important lipid second messengers (40), which provides a link of PKCµ to the recent report of sphingosine kinase activation after IgE plus Ag triggering in RBL-2H3 cells (41). If sphingosine-1-phosphate could to activate PKCµ, as has been shown for other atypical PKCs and ceramide, phosphatidic acid, and 3'phosphoinositides, the existence of a novel additionally required pathway for cytokine gene activation in mast cells would be proved.


   Acknowledgments
 
We thank P. Andrew for correcting the manuscript.


   Footnotes
 
1 Address correspondence and reprint requests to Thomas Baumruker, Novartis Research Institute, Brunner Str. 59, A-1235 Vienna, Austria. Back

2 Abbreviations used in this paper: MAPKs, mitogen-activated protein kinases; BCR, B cell receptor; AP, activator protein; PKC, protein kinase C; NF-AT, nuclear factor of activated T cells; IC50, 50% inhibitory concentration; pRL-TK, thymidine kinase promoter-dependent renilla luciferase construct; TRE, TPA-responsive element. Back

Received for publication May 23, 1997. Accepted for publication September 12, 1997.


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