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The Bcl10–Malt1 complex segregates FcɛRI-mediated nuclear factor κB activation and cytokine production from mast cell degranulation

The Bcl10–Malt1 complex segregates FcɛRI-mediated nuclear factor κB activation and cytokine... ARTICLE <doi>10.1084/jem.20051982</doi><aid>20051982</aid>The Bcl10–Malt1 complex segregates FcεRI-mediated nuclear factor κB activation and cytokine production from mast cell degranulation 1 3 4 2 Stefanie Klemm, Jan Gutermuth, Lothar Hültner, Tim Sparwasser, 3 1 5 3 Heidrun Behrendt, Christian Peschel, Tak W. Mak, Thilo Jakob, and Jürgen Ruland 1 2 Third Medical Department, Klinikum rechts der Isar and Institute for Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, 81675 Munich, Germany ZAUM-Center for Allergy and Environment, Technical University of Munich and Division of Environmental Dermatology and Allergy, GSF–National Research Center for Environment and Health, 80802 Munich, Germany Institute of Clinical Molecular Biology and Tumor Genetics, GSF–National Research Center for Environment and Health, 81377 Munich, Germany The Campbell Family Institute for Breast Cancer Research and Ontario Cancer Institute, University Health Network, University of Toronto, Toronto, Ontario, M5G 2C1, Canada Mast cells are pivotal effector cells in IgE-mediated allergic infl ammatory diseases. Central for mast cell activation are signals from the IgE receptor Fc𝛆 RI, which induce cell degranu- lation with the release of preformed mediators and de novo synthesis of proinfl ammatory leukotrienes and cytokines. How these individual mast cell responses are differentially controlled is still unresolved. We identify B cell lymphoma 10 (Bcl10) and mucosa- associated lymphoid tissue 1 (Malt1) as novel key regulators of mast cell signaling. Mice defi cient for either protein display severely impaired IgE-dependent late phase anaphylactic reactions. Mast cells from these animals neither activate nuclear factor 𝛋 B (NF-𝛋 B) nor produce tumor necrosis factor 𝛂 or interleukin 6 upon Fc𝛆 RI ligation even though proximal signaling, degranulation, and leukotriene secretion are normal. Thus, Bcl10 and Malt1 are essential positive mediators of Fc𝛆 RI-dependent mast cell activation that selectively uncouple NF-𝛋 B–induced proinfl ammatory cytokine production from degranulation and leukotriene synthesis. Mast cells are derived from myeloid progenitor for IgE, FcεRI (2, 3). Cross-linking of FcεRI- CORRESPONDENCE CORRESPONDENCE Jürgen Ruland: Jürgen Ruland: cells and widely distributed throughout vascu- bound IgE with multivalent antigen or aller- [email protected] [email protected] larized tissues. They participate in innate and gen triggers a series of biochemical events that adaptive immune defenses against bacteria and culminate in mast cell eff ector function. Sig- Abbreviations used: Bcl10, B Abbreviations used: Bcl10, B cell lymphoma 10; BCR, B cell cell lymphoma 10; BCR, B cell parasites and play a key role in IgE-mediated naling is initiated through the phosphorylation receptor; BMMC, bone mar- receptor; BMMC, bone mar- allergic diseases such as atopy and asthma, of immunoreceptor tyrosine-based activation row–derived mast cell; DNFB, row–derived mast cell; DNFB, which are responsible for increasing global motifs in the tails of the FcεRI β and γ sub- dinitrofl dinitrofl uorobenzene; uorobenzene; DNP, DNP, health problems (1). Moreover, mast cells con- units by Src family protein tyrosine kinases (2). dinitrophenyl; Erk, extracellular dinitrophenyl; Erk, extracellular signal–regulated kinase; I signal–regulated kinase; Iκ κB, B, tribute to autoimmunity and are involved in The tyrosine-phosphorylated immunoreceptor inhibitor of inhibitor of κ κB; IKK, I B; IKK, Iκ κB B pathological tissue remodeling processes that tyrosine-based activation motifs recruit the ki- kinase; Iono, ionomycin; Jnk, kinase; Iono, ionomycin; Jnk, are associated with chronic infl ammation. All nase Syk, which, together with the activated c-Jun NH c-Jun NH -terminal kinase; -terminal kinase; 2 2 Malt1, mucosa-associated lym- Malt1, mucosa-associated lym- these biological and pathological functions are receptor-proximal Src protein tyrosine kinases, phoid tissue 1; MAP, mitogen- phoid tissue 1; MAP, mitogen- triggered by mast cell–derived proinfl amma- mediates phosphorylation and consequent re- activated protein; PCA, passive activated protein; PCA, passive tory mediators such as histamine, arachidonic organization of adaptor and scaff olding proteins cutaneous anaphylaxis; PKC, cutaneous anaphylaxis; PKC, protein kinase C; SCF, stem protein kinase C; SCF, stem acid metabolites, and cytokines, which are re- at the activated FcεRI complex. Collectively, cell factor. cell factor. leased upon mast cell activation. early signaling induces the activation of down- The major stimulus for mast cell activation stream enzymes such as phosphatidylinositol is the aggregation of the high-affi nity receptor 3–kinase and phospholipase C, the generation JEM © The Rockefeller University Press $8.00 337 Vol. 203, No. 2, February 20, 2006 337–347 www.jem.org/cgi/doi/10.1084/jem.20051982 The Journal of Experimental Medicine of second messengers (e.g., inositol-1,4,5-triphosphate, 1,2- during neurodevelopment (16–20). Immunological functions diacylglycerol, and free calcium), and activation of protein of Bcl10 and Malt1 in nonlymphoid cells are still largely un- kinase C (PKC) isoforms. defi ned, and it is as of yet unknown whether they play any Ultimately, FcεRI aggregation activates several down- role in mast cells. stream pathways that initiate the allergic infl ammatory pro- In this paper, we show that Bcl10 and Malt1 are essential cess by eliciting mast cell degranulation with a rapid release of for normal mast cell function in vivo and in vitro. Although preformed vasoactive amines such as histamine and serotonin mice defi cient for either molecule exhibit normal numbers of and by triggering the de novo synthesis of proinfl ammatory skin mast cells and regular IgE-mediated immediate phase arachidonic acid metabolites and potent cytokines like TNF- anaphylactic reactions, late phase anaphylactic reactions are α or IL-6 (1). In addition, signals from the FcεRI activate severely blunted in these animals. In vitro bone marrow–de- genetic survival programs that block cell death after IgE stim- rived mast cells from Bcl10- or Malt1-defi cient mice neither ulation (4, 5). Crucial for immediate-type allergic reactions is produce TNF-α nor IL-6 in response to FcεRI stimulation, the instant degranulation, whereas mast cell–mediated late whereas the capacity to produce leukotrienes and degranula- phase reactions and IgE-induced chronic allergic infl amma- tion is normal. On a molecular level, we fi nd that FcεRI- tory processes are mainly dependent on the production of proximal signaling (the activation of the MAP kinases cytokines and the initiation of leukocyte eff ector cascades extracellular signal–regulated kinase [Erk], Jnk, and p38) and (1, 6). Major questions in mast cell biology are how early sig- the activation of Akt are intact in Bcl10- or Malt1-defi cient naling events after FcεRI aggregation are integrated and how mast cells. However, NF-κB cannot be activated in response selected mast cell responses—such as the immediate degranu- to FcεRI aggregation. Thus, we identify Bcl10 and Malt1 as lation or the delayed cytokine production—are individually crucial positive regulators of FcεRI-dependent NF-κB acti- controlled, because the identifi cation of molecules that regu- vation that selectively uncouple proinfl ammatory cytokine late specifi c mast cell eff ector functions selectively would production from degranulation and lipid mediator synthesis. provide novel targets for rational therapies of mast cell–medi- ated diseases (3). RESULTS NF-κB is a master transcription factor that controls the expression of proinfl ammatory gene products in cells of many Mast cell development is not affected by Bcl10 diff erent lineages (7). The predominant NF-κB dimer in or Malt1 defi ciency To study potential roles for Bcl10 and Malt1 in mast cells, we many cell types, including mast cells, is a p50/RelA hetero- dimer (7, 8). The activity of NF-κB is tightly controlled by fi rst assessed the eff ects of genetic Bcl10 or Malt1 disruptions inhibitor of κB (IκB) proteins that can bind to NF-κB dimers in mice on several aspects of mast cell development in vivo and in vitro (Fig. 1). Mast cells in the skin of WT, Bcl10- and retain them in an inactive state in the cytoplasm. NF-κB −/− −/− can be activated through either the canonical or the alterna- defi cient (Bcl10 ), and Malt1-defi cient (Malt1 ) mice tive pathway (7). The canonical pathway is responsible for were comparable in their morphology and anatomical distri- bution (Fig. 1 A). The frequencies of these cells in the dermis the activation of p50/RelA dimers and involves the activa- of the ear, neck, and groin were similar in all three genotypes tion of the multisubunit IκB kinase (IKK) that phosphory- lates IκB proteins on conserved serine residues to target them as determined after toluidine blue staining (Fig. 1 B). −/− −/− to ubiquitin-dependent degradation. This process frees NF- In vitro culture of WT, Bcl10 , and Malt1 bone marrow cell suspensions in the presence of IL-3 and stem cell κB and allows its translocation into the nucleus and transacti- vation of target genes. Many of the proinfl ammatory cytokine factor (SCF) revealed highly pure mast cell populations in all genes that are expressed in activated mast cells are regulated three genotypes (bone marrow–derived mast cells [BMMCs]). These cells were indistinguishable in morphology when by NF-κB (7–14). In particular, the production of TNF-α and IL-6 in response to FcεRI ligation is strictly dependent stained with toluidine blue or with alcian blue (unpublished on IKK and NF-κB activity (10, 14). Both cytokines play data). The growth rate and total cell numbers in these cul- tures, as well as the frequency of BMMCs (constantly >95%) key roles in mast cell–mediated infl ammatory responses. Yet, as revealed by fl ow cytometric analysis of the surface expres- the signaling intermediates that connect FcεRI-proximal events to IKK activation are unknown. sion of c-kit and FcεRI, were also equal (Fig. 1 C). Impor- Recently, the caspase recruitment domain protein B cell tantly, the FcεRI expression level was not infl uenced by either the Bcl10 or Malt1 disruption. Western blot analysis lymphoma 10 (Bcl10) and the paracaspase mucosa-associated lymphoid tissue 1 (Malt1) were identifi ed as key regulators of demonstrated that WT mast cells express both Bcl10 and −/− T cell and B cell antigen receptor signaling (15). Bcl10 and Malt1 (Fig. 1 D). As expected, Bcl10 mast cells do not −/− produce the Bcl10 protein, whereas Malt1 mast cells do Malt1 can directly bind to each other, and the two proteins cooperate in the assembly of a cellular complex that can me- not contain the Malt1 protein. Collectively, these fi ndings diate signal-specifi c activation of IKK. Both Bcl10 and Malt1 demonstrate that, although Bcl10 and Malt1 are expressed in normal mast cells, their absence aff ects neither skin mast cell additionally regulate the c-Jun NH -terminal kinase (Jnk) development in vivo nor BMMC diff erentiation in vitro. and p38 mitogen-activated protein (MAP) kinase pathways in lymphocytes, and Bcl10 also has a Malt1-independent role Interestingly, the expression level of Bcl10 is reduced in 338 REGULATION OF FCεRI SIGNALING BY BCL10 AND MALT1 | Klemm et al. ARTICLE We addressed the immediate and the late phase PCA re- sponses separately (Fig. 2). For early phase PCA reactions, mice were primed by in- tradermal injection of monoclonal anti-dinitrophenyl (anti- DNP) IgE antibody into the ear. 24 h later, the animals were i.v. coinjected with Evans blue dye and the antigen DNP coupled to HSA (DNP–HSA). The extravasation of Evans blue dye during the fi rst hour of the PCA reaction is mainly dependent on the degranulation of activated mast cells with rapid histamine and serotonin release resulting in locally in- creased blood vessel permeability (21). The Evans blue dye extravasation was monitored by inspection and quantifi ed 60 min after antigen challenge (Fig. 2, A and B). Neither the extravasation kinetics nor the total amount of Evans blue dye in the ear were substantially diff erent in the three genotypes, indicating that the mast cell–mediated immediate phase PCA reaction does not require Bcl10 or Malt1. We next examined the late phase PCA response that is promoted by mast cell–derived proinfl ammatory cytokines (1, 23, 24). Mice were sensitized i.v. with anti-DNP IgE an- tibody. 24 h later, the hapten dinitrofl uorobenzene (DNFB; 0.2% wt/vol) was epicutaneously applied to both sides of the ears. Although WT mice exhibited the anticipated promi- nent edema as early as 6 h and reached a maximum 24 h after −/− −/− antigen stimulation, both Bcl10 and Malt1 mice showed only a minor response (Fig. 2 C). Even 24 and 48 h after antigen challenge, the increase in ear thickness in the mutant animals reached <20% of that observed in WT mice. Therefore, we conclude that both Bcl10 and Malt1 are es- sentially required for regular IgE-dependent late phase PCA reactions in vivo. Figure 1. Mast cell development in the absence of Bcl10 or Malt1. Bcl10 and Malt1 differentially regulate Fc𝛆 RI-mediated −/− −/− (A and B) Skin biopsies from WT, Bcl10 , and Malt1 mice. (A) Repre- degranulation, lipid mediator secretion, sentative areas from the ear skin show the presence of toluidine blue– and cytokine production positive mast cells in all three genotypes (arrowheads) at 20×. Insets at To provide a basis for the defective PCA reactions in Bcl10- 100× show the boxed mast cells from each section. Bars: (full images) 10 μm; (insets) 2 μm. (B) Mast cell frequencies in skin sections from the and Malt1-defi cient mice, we examined the capacities of −/− −/− ear, neck, and groin (n = 3 mice/genotype). There was no important dif- Bcl10 , Malt1 , and WT mast cells to degranulate, syn- ference among the three genotypes. Data are means ± SEM. (C) Flow thesize, and secrete lipid mediators and to produce cytokines −/− −/− cytometric analysis of BMMCs from WT, Bcl10 , and Malt1 mice. (Fig. 3 and see Fig. 4). These and all subsequent analyses were Percentages of FcεRI and c-kit double-positive mast cells are indicated. performed on BMMCs in vitro. Results are representative of fi ve different experiments. (D) Bcl10 and −/− −/− WT, Bcl10 , and Malt1 BMMCs were loaded with −/− Malt1 expression levels in BMMCs. Western blots of WT, Bcl10 , and −/− antigen-specifi c IgE and subsequently activated by FcεRI Malt1 BMMCs with antibodies against Bcl10, Malt1, and β-actin. cross-linking with increasing doses of multivalent antigen to Results are representative of fi ve independent experiments. induce cell degranulation (Fig. 3 A). Mast cells release the enzyme β-hexosaminidase from intracellular granules whose −/− Malt1 mast cells and the Malt1 protein concentration is activity in the culture supernatant can be quantifi ed to deter- −/− smaller in Bcl10 BMMCs compared with the WT, sug- mine the effi ciency of mast cell degranulation (25). Consis- gesting that cellular levels of each of the two binding partners tent with the normal Evans blue dye extravasation during the depend on the presence of the other. immediate phase PCA reaction in vivo, mast cells derived from Bcl10- or Malt1-defi cient mice released similar amounts of β-hexosaminidase as WT BMMCs in response to FcεRI Bcl10 and Malt1 are required for normal mast cell function in vivo ligation. We also stimulated cells with PMA together with To test whether Bcl10 or Malt1 play a functional role in mast the calcium ionophore ionomycin (Iono). These pharmaco- cells in vivo, we performed two types of FcεRI-mediated logical agents bypass FcεRI-proximal signaling events and passive cutaneous anaphylaxis (PCA) experiments (21, 22). stimulate cells directly by mobilizing free calcium ions and JEM VOL. 203, February 20, 2006 339 Figure 2. Normal immediate but defective late phase PCA in statistically signifi cant ( P ≥ 0.05). (C) Late phase PCA reactions. WT, −/− −/− −/− −/− Bcl10 and Malt1 mice. (A and B) Immediate phase PCA reactions. Bcl10 , and Malt1 mice were passively sensitized by i.v. injection of Mice were passively sensitized by intradermal injection of anti-DNP IgE anti-DNP IgE. 24 h later, they were challenged by epicuta-neous applica- into the left ear. The right ear was injected with PBS. Subsequently, mice tion of DNFB to both ears, and ear thickness was measured over time. were challenged by i.v. injection of antigen in PBS/Evans blue. (A) Ears The increase in ear thickness was calculated as described in Material and −/− −/− from WT, Bcl10 , and Malt1 mice 1 h after antigen challenge. (B) The methods. Results are representative of three independent experiments. IgE-mediated Evans blue extravasation was calculated as the difference in Data are presented as means ± SEM. * and **, P ≤ 0.05 and P ≤ 0.02 −/− −/− the amount of extravasated dye in the IgE-sensitized and the nonsensi- between WT and Bcl10 or WT and Malt mice, respectively. tized control ears. Differences between the three geno-types were not −/− −/− activating PKC enzymes. Again, no substantial diff erences in of stimulation. In sharp contrast, Bcl10 and Malt1 β-hexosaminidase activity were detected in the supernatants BMMCs produced only minute amounts of TNF-α or IL-6 of BMMCs of the three genotypes (Fig. 3 A). even at later time points. To further characterize the defects Furthermore, we investigated the production of proin- in TNF-α and IL-6 production in Bcl10- and Malt1-defi - −/− −/− fl ammatory lipid mediators in WT, Bcl10 , and Malt1 cient BMMCs, we analyzed cytokine mRNA levels before BMMCs in response to FcεRI ligation using an enzyme- and 30 min after FcεRI ligation using semiquantitative RT- linked immunoassay (Fig. 3 B). Mast cells of the three geno- PCR. Unstimulated and stimulated populations of all three types produced equal amounts of the leukotrienes C , D , cell lines contained equal amounts of β-actin transcripts, indi- 4 4 and E (LTC , LTD , and LTE ). BMMC activation with cating appropriate normalization (Fig. 4 B). In WT BMMCs, 4 4 4 4 PMA/Iono also induces robust leukotriene production. both TNF-α and IL-6 mRNA levels increased substantially −/− Again, no diff erences were found between WT, Bcl10 , upon FcεRI stimulation. However, in the absence of Bcl10 −/− and Malt1 BMMCs (Fig. 3 B). Collectively, these fi rst sets or Malt1 the induction of these cytokine transcripts was ei- of in vitro experiments show that the signaling proteins Bcl10 ther absent or largely reduced compared with the WT. Thus, and Malt1 are dispensable for mast cell degranulation or leu- Bcl10 and Malt1 are critically required for FcεRI-mediated kotriene synthesis in response to FcεRI ligation or PMA/ TNF-α and IL-6 gene transcription and subsequent protein Iono treatment. production. These results indicate that the defective PCA re- It is known that the late phase PCA response in vivo is at actions observed in Bcl10- or Malt1-defi cient mice in vivo least in part induced by proinfl ammatory cytokines, in partic- might refl ect the essential requirements for the two signaling ular TNF-α, released from activated mast cells (1, 23, 24). proteins for FcεRI-mediated cytokine production. Therefore, we were especially interested in measuring the transcriptional induction, production, and release of cyto- Bcl10 and Malt1 are dispensable for IgE-induced mast −/− −/− kines by Bcl10 and Malt1 mast cells in response to cell survival FcεRI stimulation in vitro. To this end, we fi rst performed In lymphocytes, Bcl10 and Malt1 are not only vital for cell time course experiments and analyzed the concentrations of activation but also involved in the regulation of cell survival TNF-α and IL-6 in the supernatant of stimulated cells by (15). Because recent studies demonstrated a role for FcεRI- ELISA. WT mast cells produced both mediators readily (Fig. derived signals in the control of mast cell survival (4, 5), we 4 A). The maximal concentrations were detected after 2 h also studied the contributions of Bcl10 and Malt1 to this 340 REGULATION OF FCεRI SIGNALING BY BCL10 AND MALT1 | Klemm et al. ARTICLE Figure 3. Normal degranulation and leukotriene synthesis in −/− −/− −/− Bcl10 and Malt1 BMMCs. (A) Regular degranulation of Bcl10 −/− −/− −/− and Malt1 mast cells. BMMCs from WT, Bcl10 , and Malt mice were loaded with anti-DNP IgE and subsequently stimulated for 30 min with the indicated concentrations of DNP–HSA. Alternatively, BMMCs were left unsensitized and stimulated with PMA/Iono (100 nM each) for 30 min. The extent of degranulation was determined by measuring the activity of the granular enzyme β-hexosaminidase in supernatants and cell lysates and calculated as described in Material and methods. Data are means ± SEM from triplicate samples and are representative of four in- −/− dependent experiments. (B) Regular leukotriene synthesis in Bcl10 and −/− Malt1 mast cells. Anti-DNP IgE-sensitized BMMCs from all three geno- types were stimulated with DNP–HSA at the indicated concentrations, or −/− −/− Figure 4. Defective cytokine production in Bcl10 and Malt1 BMMCs were left unsensitized and stimulated with PMA/Iono (100 nM −/− −/− BMMCs. (A) Impaired cytokine secretion in Bcl10 and Malt1 mast each). 60 min later, the supernatants of the stimulated cells were col- −/− −/− cells. WT, Bcl10 , and Malt1 BMMCs were loaded with anti-DNP IgE lected and assayed for the concentrations of LTC , LTD , and LTE . Data 4 4 4 and stimulated with DNP–HSA for the indicated intervals. Supernatants are means ± SEM from triplicate samples and representative of four were collected, and TNF-α and IL-6 protein concentrations were deter- independent experiments. mined by ELISA. Data are means ± SEM from triplicate samples and rep- resentative of at least fi ve independent experiments. * and **, P ≤ 0.05 −/− −/− and P ≤ 0.01 between WT and Bcl10 or WT and Malt1 BMMCs, −/− −/− −/− pathway (Fig. 5). BMMCs of WT, Bcl10 , and Malt1 respectively. (B) Defective expression of cytokine mRNAs in Bcl10 and −/− −/− −/− Malt1 mast cells. BMMCs from WT, Bcl10 , and Malt1 mice were mice were incubated for 4 d in media with low concentra- stimulated for 30 min as in A. RNA was purifi ed, and TNF-α, IL-6, and tions of FCS without IL-3 and SCF. This growth factor −/− β-actin mRNAs were detected by semiquantitative RT-PCR using specifi c withdrawal induced apoptotic death of WT, Bcl10 , and primers. Data are representative of three independent experiments. −/− Malt1 BMMCs. The frequency of apoptotic cells was in- dependent of the presence of Bcl10 or Malt1 as determined by fl ow cytometry after annexin V/propidium iodide stain- ing. (Fig. 5 A). Stimulation of cells with IgE alone or in com- defi cient mast cells, we analyzed the signaling pathways bination with antigen (unpublished data) rescued cell death downstream of FcεRI stimulation (Fig. 6 and see Fig. 7). and up-regulated the expression of the prosurvival protein Ligation of IgE-occupied FcεRI molecules with antigen Bcl-X in mast cells of all three genotypes similarly (Fig. 5 B). results in the rapid activation of receptor-proximal tyrosine We thus conclude that Bcl10 and Malt1 are not essential for kinases, which is a prerequisite for all mast cell eff ector func- the FcεRI-controlled survival pathway. tions (2). Consistent with the normal degranulation and lipid −/− −/− mediator synthesis in Bcl10 and Malt1 mast cells, re- Bcl10 and Malt1 are essential regulators ceptor-proximal signaling does not involve Bcl10 or Malt1 of Fc𝛆 RI-controlled NF-𝛋 B activation because the tyrosine phosphorylation patterns induced by To defi ne the molecular mechanisms responsible for the FcεRI stimulation did not diff er in kinetics or intensity defective cytokine gene expression in Bcl10- and Malt1- among BMMCs of the three genotypes (Fig. 6 A). JEM VOL. 203, February 20, 2006 341 mediated NF-κB activation. To test this hypothesis, we analyzed IκB-α phosphorylation and its subsequent degra- dation in FcεRI-stimulated BMMCs of the three geno- types. FcεRI signaling in WT cells induced rapid IκB-α phosphorylation and, consequently, its degradation (Fig. 7 A). In contrast, signaling to IκB-α was completely abol- −/− −/− ished in Bcl10 or in Malt1 mast cells. Direct PKC activation via PMA/Iono stimulation also resulted in IκB-α phosphorylation and degradation only in the WT cells but not in BMMCs defi cient for either Bcl10 or Malt1, indi- cating a requirement for Bcl10 and Malt1 downstream of PKC activation. To directly investigate NF-κB nuclear translocation and DNA binding, we performed gel shift assays with nuclear ex- tracts from stimulated mast cells. Consistent with the marked impairment of IκB-α phosphorylation and degradation in −/− −/− Bcl10 and Malt1 BMMCs, NF-κB was only activated in WT mast cells but not in those defi cient for Bcl10 or Malt1 (Fig. 7 B). Thus, Bcl10 and Malt1 are both essential to spe- cifi cally couple FcεRI and PKC signaling to the activation of the NF-κB pathway in mast cells. Figure 5. Regular IgE-induced survival signaling in the absence of Bcl10 or Malt1. (A) BMMCs from all three genotypes were deprived of DISCUSSION the mast cell survival cytokines IL-3 and SCF and cultured in media with In this paper, we have demonstrated on a genetic basis that low concentrations of FCS (1%) with or without 5 μg/ml anti-DNP IgE Bcl10 and Malt1 are essential and nonredundant positive reg- mAb. 48 and 96 h later, apoptotic cell death was determined by fl ow ulators of mast cell activation and eff ector function. Although cytometry after annexin V/propidium iodide staining. The percentages of mast cells seem to diff erentiate and survive normally in the viable mast cells are shown. Data are means ± SEM from triplicate sam- ples and are representative of four independent experiments. No impor- absence of either Bcl10 or Malt1, both proteins are critically tant differences were detected among the three genotypes. (B) BMMCs required for NF-κB activation induced by the high-affi nity were cultured as in A and left unstimulated or stimulated for 24 h with IgE receptor FcεRI. Bcl10- or Malt1-defi cient mast cells do 5 μg/ml anti-DNP IgE. Total cell lysates were resolved by SDS-PAGE and not produce the NF-κB–regulated proinfl ammatory cyto- immunoblotted with anti–Bcl-X antibody and, after stripping, reprobed kines TNF-α and IL-6 and, consistently, late phase PCA re- with anti–β-actin antibody to control for equal loading. actions are severely impaired in Bcl10- or Malt1-defi cient mice in vivo. Activation of mast cells through the FcεRI initiates a FcεRI-proximal signaling activates the Erk, Jnk, and cascade of events that lead to degranulation, production of p38 pathways, as well as the phosphatidylinositol 3–kinase lipid mediators such as leukotrienes, and transcriptional up- pathway that results in the activation of the serine kinase regulation and release of proinfl ammatory cytokines such as Akt. These cascades regulate both cytokine production and TNF-α or IL-6. These all represent normal mast cell func- generation of arachidonic acid metabolites upon FcεRI li- tions that, depending on the context, may be benefi cial or gation (1, 2). Bcl10 and Malt1 have been implicated in the harmful in the setting of innate or allergic immune responses. regulation of Jnk and p38 signaling in lymphocytes (18, 19). Bcl10 and Malt1 are not involved in FcεRI-proximal signal- However, using immunoblotting with activation state–spe- ing events that are known to lead to the activation of PKC cifi c phosphoantibodies against Jnk, p38, the p42 and p44 enzymes (2). The PKC family consists of at least 10 serine/ MAP kinases Erk1 and Erk2, or Akt, we could not detect threonine kinases, and previous studies have indicated that diff erences in the FcεRI-induced activation of any of these PKC activation is involved in all aspects of mast cell eff ector −/− −/− pathways among WT, Bcl10 , and Malt1 mast cells functions (26). Although comprehensive studies of the indi- (Fig. 6 B). We additionally activated the cells with PMA/ vidual roles of all PKC isoforms in mast cell biology are Iono and again detected similar activation for Jnk, p38, missing, chemical or genetic inhibition of the PMA- and cal- Erk1, Erk2, and Akt in the presence or absence of either cium-sensitive conventional isoforms PKCα and PKCβ Bcl10 or Malt1 (Fig. 6 C). identifi ed these kinases as essential inducers of degranulation As earlier mentioned, the expression of TNF-α and IL- and cytokine and leukotriene production (27–29). The 6 critically depends on the activation of NF-κB. Based on PMA-sensitive but calcium-insensitive isoform PKCδ is in- known functions of Bcl10 and Malt1 in lymphocytes and volved in the activation of leukotriene synthesis (30) and can their newly discovered requirement in mast cells, we rea- simultaneously function as a negative regulator of antigen- soned that the two proteins might be involved in FcεRI- induced degranulation (31). 342 REGULATION OF FCεRI SIGNALING BY BCL10 AND MALT1 | Klemm et al. ARTICLE In this paper, we show that FcεRI- and PMA/Iono-in- duced IκB-α phosphorylation, degradation, and NF-κB ac- tivation are completely abolished in Bcl10- or Malt1-defi cient mast cells. These results reveal an essential requirement for Bcl10–Malt1 signaling downstream of the FcεRI and down- stream of all PMA-responsive PKCs in mast cells. Impor- tantly, however, FcεRI- or PMA/Iono-induced activation −/− of the Jnk, p38, Erk, and Akt pathways are intact in Bcl10 −/− and Malt1 mast cells, indicating that the Bcl10–Malt1 complex does not aff ect all PKC downstream pathways. Consistent with the regular activation of selected FcεRI or PKC eff ector signals, we observed normal mast cell survival, degranulation, and leukotriene production in the absence of Bcl10 or Malt1. The observation that TNF-α and IL-6 transcription and protein production are severely impaired in Bcl10- or Malt1- defi cient mast cells is in agreement with our fi ndings that Bcl10–Malt1 specifi cally controls FcεRI- induced NF-κB activation and the known role of NF-κB for cytokine gene expression. Considering all in vitro results collectively, we propose a model for the molecular func- tions of Bcl10 and Malt1 in mast cell signaling that is de- picted in Fig. 7 C. This signaling model is consistent with the in vivo observations in Bcl10- and Malt1-defi cient mice, as immediate phase PCA reactions that depend on mast cell degranulation (21) are normal, whereas late phase reactions that are at least partially dependent on transcriptional induc- tion and the release of mast cell–derived cytokines (1, 24) are severely impaired in the absence of either Bcl10 or Malt1. However, because in the mutant animals all cells lack Bcl10 or Malt1, signaling defi ciencies in other cells besides mast cells could additionally contribute to the defective late phase PCA response. Experiments with genetically altered mice have recently elucidated many aspects of positive and negative regulation of FcεRI-induced mast cell activation (1). Collectively, these fi ndings demonstrate that antigen receptor–specifi c mole- cules required for FcεRI signaling generally aff ect several as- pects of mast cell function simultaneously. In contrast, we identifi ed the Bcl10–Malt1 signaling complex as a molecular structure that selectively regulates one downstream eff ector arm of FcεRI, namely NF-κB–mediated cytokine produc- tion, but does not aff ect mast cell survival, degranulation, or the synthesis of leukotrienes. The conclusion that the Bcl10–Malt1 complex operates selectively downstream of PKCs in FcεRI signaling to NF-κB are shown on the left. Data are representative of three independent ex- periments. (B) Immunoblot with anti–phospho-p38, anti–phospho- Figure 6. Normal Fc𝛆 RI proximal signaling and regular p38, Erk, p44/42, anti–phospho-Jnk, and anti–phospho-Akt. After stripping, −/− −/− Jnk, and Akt activation in Bcl10 and Malt1 mast cells. membranes were reprobed with anti-p38, anti-p44/p42, anti-Jnk, and −/− −/− (A and B) WT, Bcl10 , and Malt1 BMMCs sensitized with anti-DNP anti-Akt antibody, respectively. Data are representative of at least three −/− −/− IgE were stimulated with DNP–HSA for the indicated time intervals (in independent experiments. (C) Unsensitized WT, Bcl10 , and Malt1 minutes) as described in Material and methods. Total cell lysates were BMMCs were stimulated with PMA/Iono for the indicated time intervals separated by SDS-PAGE (2 × 10 cell equivalents). (A) Immunoblot (in minutes) as described in Material and methods and immunoblotted as with anti-phosphotyrosine antibodies. Molecular mass (kD) markers in B. Results are representative of three independent experiments. JEM VOL. 203, February 20, 2006 343 Figure 7. Bcl10 and Malt1 are required for NF-𝛋 B signaling in PMA/Iono (100 nM each) for 60 min. Subsequently, nuclear extracts were mast cells. (A) Defective IκB-α phosphorylation and degradation after prepared and subjected to gel mobility shift assays using a radiolabeled −/− −/− FcεRI ligation or PMA/Iono treatment in Bcl10 or Malt1 mast cells. probe containing NF-κB binding site sequences. Results are representa- BMMCs from all genotypes were sensitized with anti-DNP IgE and stimu- tive of three independent experiments. (C) A model for the role of Bcl10 lated with DNP–HSA for the indicated time intervals (in minutes). Alterna- and Malt1 in FcεRI-mediated signal transduction after FcεRI receptor tively, cells were left unsensitized and stimulated with PMA/Iono (100 nM stimulation, Bcl10 and Malt1 operate downstream of PKCs and serve each). IκB-α phosphorylation and degradation were determined by West- as selective signal transducers that activate the IκB-dependent NF-κB ern blotting. Membranes were reprobed with anti–β-actin antibody to pathway. The Bcl10–Malt1 complex selectively controls proinfl ammatory control for equal loading. Data are representative of at least four inde- cytokine production but does not regulate mast cell degranulation or −/− pendent experiments. (B) Defective NF-κB activation in Bcl10 and leukotriene synthesis. For details, see Discussion. −/− Malt1 mast cells. BMMCs were left unstimulated or stimulated with induced NF-κB activation but selectively controls p38 MAP provides novel insights into context-specifi c immune regu- lation. The FcεRI on mast cells belongs to the family of kinase signaling in response to BCR ligation (40). Our fi nd- multisubunit immunoreceptors that also includes the TCR ings that FcεRI signaling requires both Bcl10 and Malt1 for and the B cell receptor (BCR), but numerous genetic stud- NF-κB control but not for Jnk or p38 activation extend this ies have demonstrated that each of these receptors uses idea of receptor-specifi c utilization of the signaling module unique sets of downstream molecules to diff erentially medi- and reinforces the necessity to investigate the precise func- ate cell activation. For example, the FcεRI requires the ty- tion of each signal transducer in distinct cell lineages. So far rosine kinases Syk and Btk and the adaptor molecule linker there are no other reports available that explore roles of for activation of T cells to mediate mast cell activation (32– Bcl10, Malt1, or any of their binding partners in FcεRI sig- 34). Syk and Btk are additionally required for BCR signal- naling. It will thus be additionally important to study mast cells in mice defi cient in Carma proteins, TNF receptor– ing but are completely dispensable for T cell activation, whereas the linker for activation of T cells is essential for associated factor molecules, and MAP kinase–activating ki- TCR signaling but has no role in B cells (35–37). The nases, including TAK1, to understand precisely how the Bcl10–Malt1 complex is also known to be diff erentially used FcεRI diff erentially couples to NF-κB and Jnk/p38 down- by immunoreceptors. Bcl10 can bind to Carma molecules stream signaling. These studies could also reveal the Bcl10– and recruit Malt1 and various TNF receptor–associated fac- Malt1–independent mechanisms of Jnk and p38 activation tor molecules and kinases such as TAK1 into signaling com- and give further insights into the selective control of distinct plexes to mediate downstream activation of NF-κB, Jnk, mast cell eff ector functions. and p38 (15, 19, 38–40). TCR signaling to NF-κB critically Recent biochemical studies have shown that Bcl10–Malt1 depends on Bcl10, Malt1, and the kinase TAK1 (39). How- signaling to IKK and NF-κB involves the oligomerization of Bcl10 and Malt1 and subsequent Malt1-dependent regu- ever, Malt1 is partially expendable for BCR signaling to NF-κB (18), and TAK1 is completely dispensable for BCR- latory lysine-63–linked ubiquitinylation of IKKγ/NEMO, 344 REGULATION OF FCεRI SIGNALING BY BCL10 AND MALT1 | Klemm et al. ARTICLE Mast cell degranulation. To induce degranulation, 2 × 10 BMMCs/ml which induces IKK activity (39, 41). These fi ndings open were loaded with 5 μg/ml anti-DNP IgE mAb (SPE-7; Sigma-Aldrich) for the possibility that Bcl10–Malt1 signaling could potentially 1 h on ice in Tyrode’s buff er (10 mM Hepes, pH 7.4, 130 mM NaCl, 5 mM be inhibited by targeted drugs because numerous kinase KCl, 1.4 mM CaCl , 1 mM MgCl , 5.6 mM glucose, and 0.1% BSA). After 2 2 inhibitors and many compounds that target the ubiquitin/ washing, sensitized cells were stimulated with the concentrations of DNP– proteasome system are in preclinical or clinical development. HSA indicated in the fi gures. Alternatively, cells were left unsensitized and Mast cell–derived TNF-α recruits and activates neutrophils stimulated with 100 nM PMA (Sigma-Aldrich) and 100 nM Iono (Sigma- Aldrich) in Tyrode’s buff er. The enzymatic activities of β-hexosaminidase in and lymphocytes at sites of infl ammation and has been impli- supernatants and cell pellets solubilized with 0.5% Triton X-100 in Tyrode’s cated in tissue remodeling processes, angiogenesis, and fi bro- Buff er were measured with p-nitrophenyl-N-acetyl-β-d-glucosaminide sis that can be so prominent in IgE-associated chronic allergic (Sigma-Aldrich), and the percentage of degranulation was calculated as pre- diseases (1, 23, 42, 43). Consequently, there is a tremendous viously described (25). interest in inhibiting TNF-α production in allergic infl am- mation. In this paper, we demonstrate that a complete ge- 6 Measurements of leukotrienes and cytokines. 2 × 10 BMMCs/ml netic blockage of Bcl10–Malt1 signaling thoroughly abolishes were loaded with 0.5 μg/ml anti-DNP IgE mAb (SPE-7) overnight, washed twice, and stimulated with DNP–HSA or left unsensitized and stimulated FcεRI-specifi c NF-κB activation and TNF-α and IL-6 pro- with PMA and Iono (100 nM each) as indicated in the fi gures. Cell superna- duction in mast cells. Thus, we suggest that specifi c thera- tants were harvested, and leukotrienes LTC , LTD , and LTE were deter- 4 4 4 peutic targeting of Bcl10–Malt1 signaling could potentially mined by enzyme-linked immunoassay (GE Healthcare), whereas IL-6 and contribute to the therapy of mast cell–mediated infl amma- TNF-α were determined by ELISA DuoSets (R&D Systems), as recom- tory diseases without the toxic side eff ects that can be ex- mended by the manufacturer. pected from general NF-κB inhibition. RT-PCR. 2 × 10 BMMCs/ml were loaded with 0.5 μg/ml anti-DNP IgE mAb (SPE-7) overnight, washed twice, and stimulated with 20 ng/ml MATERIALS AND METHODS DNP–HSA for 30 min. Total RNA was isolated using TRIzol reagent Mice. The generation of Bcl10- and Malt1-defi cient mice was reported (Invitrogen), according to the manufacturer’s instructions, and reverse tran- previously (17, 18). Mice were housed under specifi c pathogen-free condi- scribed using the Superscript fi rst-strand synthesis system (Invitrogen). Prim- tions. The study was conducted under federal guidelines for the use and care ers and PCR conditions were described elsewhere (25). of laboratory animals and was approved by the Government of the District of Upper Bavaria. Mice were of a C57BL/6 and 129/J mixed background. Survival assay. BMMCs were cultured as indicated in the fi gures. Cell via- Littermates were used in each experiment. bility was quantifi ed by fl ow cytometry after annexin V/propidium iodide staining as recommended by the manufacturer (BD Biosciences). Skin histology. Naive animals at 10–12 wk of age were killed, and skin samples from the ear, neck, and groin regions were removed and fi xed in 4% Signal transduction. For FcεRI signaling, 2 × 10 BMMCs/ml were formalin. 4-μm tissue sections were stained with toluidine blue, and mast preloaded with 0.5 μg/ml anti-DNP IgE mAb (SPE-7) and subsequently cells were counted in the dermis of each sample at 40×. 10 fi elds at 10× (per activated by adding 20 ng/ml DNP–HSA, as indicated in the fi gures. Alter- mouse and location) were used to delineate and integrate the dermal area natively, BMMCs were left unsensitized and stimulated with PMA and (KS 300 software; Carl Zeiss MicroImaging, Inc.) that was analyzed. Iono (100 nM each) or with 5 μg/ml anti-DNP IgE mAb for Bcl-X in- duction. Cells were lysed, and denatured proteins were separated on 10% BMMC culture and FACS analysis. Bone marrow cells were cultured in polyacrylamide gels and subjected to immunoblotting using antibodies suspension in RPMI 1640 with 20% FCS, 30 ng/ml murine IL-3 (R&D against phosphotyrosine (PY99; Santa Cruz Biotechnology, Inc.), phos- Systems), 50 ng/ml murine SCF (R&D Systems), and 150 μM monothio- pho-p38, p38, phospho-p44/42, p44/42, phospho-Jnk, Jnk, phospho-Akt, glycerol (Sigma-Aldrich). To quantify BMMC frequencies, cells were stained Akt, phospho–IκB-α, Iκb-α, Bcl-X (all from Cell Signaling), or β-actin with FITC-labeled anti-FcεRI and PE-labeled anti–c-kit antibody (eBiosci- (Sigma-Aldrich). For electromobility shift assays, 10 BMMCs/sample were ence) and analyzed by fl ow cytometry (FACScan; Becton Dickinson). stimulated with PMA/Iono, and gel shifts were performed as described previously (17). PCA. For immediate phase PCA reactions, mice were passively sensitized by intradermal injection of 250 ng anti-DNP IgE mAb (clone H1-ε-26; Statistical analysis. Results were analyzed for statistical signifi cance with provided by F.-T. Liu [University of California, Davis, Sacramento, CA] the unpaired two-tailed Student’s t test (Excel; Microsoft). Data from and D.H. Katz [Avanir Pharmaceuticals, San Diego, CA]) into one ear and −/− −/− Bcl10 and Malt1 mice or BMMCs were separately compared with the PBS injection into the contralateral ear. 24 h later, mice were challenged by WT. Diff erences between groups were considered signifi cant at P ≤ 0.05. i.v. injection of 150 μg of DNP–HSA (30–40 moles DNP/mol HSA; Sigma-Aldrich) in 100 μl Evans blue dye (0.5%; Sigma-Aldrich). To deter- We thank Michael Huber and Margot Thome for helpful discussions and advice; mine the amount of extravasated dye, mice were killed, ears were removed, Frank Redegeld for affi nity purifying H1-ε-s26 mAb; and Britta Dorn for excellent and Evans blue dye was extracted with potassium hydroxide and photomet- technical assistance. rically quantifi ed as described previously (22). This work was supported by grants from the German National Genome Project For late phase PCA reactions, mice were passively sensitized by i.v. (NUWS31T05S15T03, subproject 3b), the German Federal Ministry of Science and injection of 2 μg anti-DNP IgE mAb (clone H1-ε-26) or left unsensitized. Education (BMBF 01GC0104, subproject 2), and the Technical University of Munich 24 h later, sensitized and nonsensitized mice were challenged by epicutane- Medical School (KKF 8760179) to T. Jakob and by a Max-Eder-Program grant from ous application of 10 μl of DNFB (0.2% wt/vol) in acetone/olive oil (4:1) Deutsche Krebshilfe and grants from the Deutsche Forschungsgemeinschaft (SFB to both sides of both ears. The ear swelling response was assessed by measur- 455 and SFB 456) to J. Ruland. ing the ear thickness using an engineer’s micrometer dial thickness gauge. The authors have no confl icting fi nancial interests. The increment of ear thickness (postchallenge value – prechallenge baseline value) was expressed as the percentage of the baseline value obtained before Submitted: 4 October 2005 antigen challenge. Accepted: 23 December 2005 JEM VOL. 203, February 20, 2006 345 21. Inagaki, N., S. Goto, H. Nagai, and A. Koda. 1986. Homologous pas- REFERENCES sive cutaneous anaphylaxis in various strains of mice. Int. Arch. Allergy 1. Galli, S.J., J. Kalesnikoff , M.A. Grimbaldeston, A.M. Piliponsky, C.M. Williams, and M. Tsai. 2005. Mast cells as “tunable” eff ector and Appl. 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The Bcl10–Malt1 complex segregates FcɛRI-mediated nuclear factor κB activation and cytokine production from mast cell degranulation

Klemm, Stefanie; Gutermuth, Jan; Hültner, Lothar; Sparwasser, Tim; Behrendt, Heidrun; Peschel, Christian; Mak, Tak W.; Jakob, Thilo; Ruland, Jürgen
The Journal of Experimental Medicine , Volume 203 (2) – Feb 20, 2006
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Copyright © 2006, The Rockefeller University Press
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0022-1007
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10.1084/jem.20051982
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Abstract

ARTICLE <doi>10.1084/jem.20051982</doi><aid>20051982</aid>The Bcl10–Malt1 complex segregates FcεRI-mediated nuclear factor κB activation and cytokine production from mast cell degranulation 1 3 4 2 Stefanie Klemm, Jan Gutermuth, Lothar Hültner, Tim Sparwasser, 3 1 5 3 Heidrun Behrendt, Christian Peschel, Tak W. Mak, Thilo Jakob, and Jürgen Ruland 1 2 Third Medical Department, Klinikum rechts der Isar and Institute for Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, 81675 Munich, Germany ZAUM-Center for Allergy and Environment, Technical University of Munich and Division of Environmental Dermatology and Allergy, GSF–National Research Center for Environment and Health, 80802 Munich, Germany Institute of Clinical Molecular Biology and Tumor Genetics, GSF–National Research Center for Environment and Health, 81377 Munich, Germany The Campbell Family Institute for Breast Cancer Research and Ontario Cancer Institute, University Health Network, University of Toronto, Toronto, Ontario, M5G 2C1, Canada Mast cells are pivotal effector cells in IgE-mediated allergic infl ammatory diseases. Central for mast cell activation are signals from the IgE receptor Fc𝛆 RI, which induce cell degranu- lation with the release of preformed mediators and de novo synthesis of proinfl ammatory leukotrienes and cytokines. How these individual mast cell responses are differentially controlled is still unresolved. We identify B cell lymphoma 10 (Bcl10) and mucosa- associated lymphoid tissue 1 (Malt1) as novel key regulators of mast cell signaling. Mice defi cient for either protein display severely impaired IgE-dependent late phase anaphylactic reactions. Mast cells from these animals neither activate nuclear factor 𝛋 B (NF-𝛋 B) nor produce tumor necrosis factor 𝛂 or interleukin 6 upon Fc𝛆 RI ligation even though proximal signaling, degranulation, and leukotriene secretion are normal. Thus, Bcl10 and Malt1 are essential positive mediators of Fc𝛆 RI-dependent mast cell activation that selectively uncouple NF-𝛋 B–induced proinfl ammatory cytokine production from degranulation and leukotriene synthesis. Mast cells are derived from myeloid progenitor for IgE, FcεRI (2, 3). Cross-linking of FcεRI- CORRESPONDENCE CORRESPONDENCE Jürgen Ruland: Jürgen Ruland: cells and widely distributed throughout vascu- bound IgE with multivalent antigen or aller- [email protected] [email protected] larized tissues. They participate in innate and gen triggers a series of biochemical events that adaptive immune defenses against bacteria and culminate in mast cell eff ector function. Sig- Abbreviations used: Bcl10, B Abbreviations used: Bcl10, B cell lymphoma 10; BCR, B cell cell lymphoma 10; BCR, B cell parasites and play a key role in IgE-mediated naling is initiated through the phosphorylation receptor; BMMC, bone mar- receptor; BMMC, bone mar- allergic diseases such as atopy and asthma, of immunoreceptor tyrosine-based activation row–derived mast cell; DNFB, row–derived mast cell; DNFB, which are responsible for increasing global motifs in the tails of the FcεRI β and γ sub- dinitrofl dinitrofl uorobenzene; uorobenzene; DNP, DNP, health problems (1). Moreover, mast cells con- units by Src family protein tyrosine kinases (2). dinitrophenyl; Erk, extracellular dinitrophenyl; Erk, extracellular signal–regulated kinase; I signal–regulated kinase; Iκ κB, B, tribute to autoimmunity and are involved in The tyrosine-phosphorylated immunoreceptor inhibitor of inhibitor of κ κB; IKK, I B; IKK, Iκ κB B pathological tissue remodeling processes that tyrosine-based activation motifs recruit the ki- kinase; Iono, ionomycin; Jnk, kinase; Iono, ionomycin; Jnk, are associated with chronic infl ammation. All nase Syk, which, together with the activated c-Jun NH c-Jun NH -terminal kinase; -terminal kinase; 2 2 Malt1, mucosa-associated lym- Malt1, mucosa-associated lym- these biological and pathological functions are receptor-proximal Src protein tyrosine kinases, phoid tissue 1; MAP, mitogen- phoid tissue 1; MAP, mitogen- triggered by mast cell–derived proinfl amma- mediates phosphorylation and consequent re- activated protein; PCA, passive activated protein; PCA, passive tory mediators such as histamine, arachidonic organization of adaptor and scaff olding proteins cutaneous anaphylaxis; PKC, cutaneous anaphylaxis; PKC, protein kinase C; SCF, stem protein kinase C; SCF, stem acid metabolites, and cytokines, which are re- at the activated FcεRI complex. Collectively, cell factor. cell factor. leased upon mast cell activation. early signaling induces the activation of down- The major stimulus for mast cell activation stream enzymes such as phosphatidylinositol is the aggregation of the high-affi nity receptor 3–kinase and phospholipase C, the generation JEM © The Rockefeller University Press $8.00 337 Vol. 203, No. 2, February 20, 2006 337–347 www.jem.org/cgi/doi/10.1084/jem.20051982 The Journal of Experimental Medicine of second messengers (e.g., inositol-1,4,5-triphosphate, 1,2- during neurodevelopment (16–20). Immunological functions diacylglycerol, and free calcium), and activation of protein of Bcl10 and Malt1 in nonlymphoid cells are still largely un- kinase C (PKC) isoforms. defi ned, and it is as of yet unknown whether they play any Ultimately, FcεRI aggregation activates several down- role in mast cells. stream pathways that initiate the allergic infl ammatory pro- In this paper, we show that Bcl10 and Malt1 are essential cess by eliciting mast cell degranulation with a rapid release of for normal mast cell function in vivo and in vitro. Although preformed vasoactive amines such as histamine and serotonin mice defi cient for either molecule exhibit normal numbers of and by triggering the de novo synthesis of proinfl ammatory skin mast cells and regular IgE-mediated immediate phase arachidonic acid metabolites and potent cytokines like TNF- anaphylactic reactions, late phase anaphylactic reactions are α or IL-6 (1). In addition, signals from the FcεRI activate severely blunted in these animals. In vitro bone marrow–de- genetic survival programs that block cell death after IgE stim- rived mast cells from Bcl10- or Malt1-defi cient mice neither ulation (4, 5). Crucial for immediate-type allergic reactions is produce TNF-α nor IL-6 in response to FcεRI stimulation, the instant degranulation, whereas mast cell–mediated late whereas the capacity to produce leukotrienes and degranula- phase reactions and IgE-induced chronic allergic infl amma- tion is normal. On a molecular level, we fi nd that FcεRI- tory processes are mainly dependent on the production of proximal signaling (the activation of the MAP kinases cytokines and the initiation of leukocyte eff ector cascades extracellular signal–regulated kinase [Erk], Jnk, and p38) and (1, 6). Major questions in mast cell biology are how early sig- the activation of Akt are intact in Bcl10- or Malt1-defi cient naling events after FcεRI aggregation are integrated and how mast cells. However, NF-κB cannot be activated in response selected mast cell responses—such as the immediate degranu- to FcεRI aggregation. Thus, we identify Bcl10 and Malt1 as lation or the delayed cytokine production—are individually crucial positive regulators of FcεRI-dependent NF-κB acti- controlled, because the identifi cation of molecules that regu- vation that selectively uncouple proinfl ammatory cytokine late specifi c mast cell eff ector functions selectively would production from degranulation and lipid mediator synthesis. provide novel targets for rational therapies of mast cell–medi- ated diseases (3). RESULTS NF-κB is a master transcription factor that controls the expression of proinfl ammatory gene products in cells of many Mast cell development is not affected by Bcl10 diff erent lineages (7). The predominant NF-κB dimer in or Malt1 defi ciency To study potential roles for Bcl10 and Malt1 in mast cells, we many cell types, including mast cells, is a p50/RelA hetero- dimer (7, 8). The activity of NF-κB is tightly controlled by fi rst assessed the eff ects of genetic Bcl10 or Malt1 disruptions inhibitor of κB (IκB) proteins that can bind to NF-κB dimers in mice on several aspects of mast cell development in vivo and in vitro (Fig. 1). Mast cells in the skin of WT, Bcl10- and retain them in an inactive state in the cytoplasm. NF-κB −/− −/− can be activated through either the canonical or the alterna- defi cient (Bcl10 ), and Malt1-defi cient (Malt1 ) mice tive pathway (7). The canonical pathway is responsible for were comparable in their morphology and anatomical distri- bution (Fig. 1 A). The frequencies of these cells in the dermis the activation of p50/RelA dimers and involves the activa- of the ear, neck, and groin were similar in all three genotypes tion of the multisubunit IκB kinase (IKK) that phosphory- lates IκB proteins on conserved serine residues to target them as determined after toluidine blue staining (Fig. 1 B). −/− −/− to ubiquitin-dependent degradation. This process frees NF- In vitro culture of WT, Bcl10 , and Malt1 bone marrow cell suspensions in the presence of IL-3 and stem cell κB and allows its translocation into the nucleus and transacti- vation of target genes. Many of the proinfl ammatory cytokine factor (SCF) revealed highly pure mast cell populations in all genes that are expressed in activated mast cells are regulated three genotypes (bone marrow–derived mast cells [BMMCs]). These cells were indistinguishable in morphology when by NF-κB (7–14). In particular, the production of TNF-α and IL-6 in response to FcεRI ligation is strictly dependent stained with toluidine blue or with alcian blue (unpublished on IKK and NF-κB activity (10, 14). Both cytokines play data). The growth rate and total cell numbers in these cul- tures, as well as the frequency of BMMCs (constantly >95%) key roles in mast cell–mediated infl ammatory responses. Yet, as revealed by fl ow cytometric analysis of the surface expres- the signaling intermediates that connect FcεRI-proximal events to IKK activation are unknown. sion of c-kit and FcεRI, were also equal (Fig. 1 C). Impor- Recently, the caspase recruitment domain protein B cell tantly, the FcεRI expression level was not infl uenced by either the Bcl10 or Malt1 disruption. Western blot analysis lymphoma 10 (Bcl10) and the paracaspase mucosa-associated lymphoid tissue 1 (Malt1) were identifi ed as key regulators of demonstrated that WT mast cells express both Bcl10 and −/− T cell and B cell antigen receptor signaling (15). Bcl10 and Malt1 (Fig. 1 D). As expected, Bcl10 mast cells do not −/− produce the Bcl10 protein, whereas Malt1 mast cells do Malt1 can directly bind to each other, and the two proteins cooperate in the assembly of a cellular complex that can me- not contain the Malt1 protein. Collectively, these fi ndings diate signal-specifi c activation of IKK. Both Bcl10 and Malt1 demonstrate that, although Bcl10 and Malt1 are expressed in normal mast cells, their absence aff ects neither skin mast cell additionally regulate the c-Jun NH -terminal kinase (Jnk) development in vivo nor BMMC diff erentiation in vitro. and p38 mitogen-activated protein (MAP) kinase pathways in lymphocytes, and Bcl10 also has a Malt1-independent role Interestingly, the expression level of Bcl10 is reduced in 338 REGULATION OF FCεRI SIGNALING BY BCL10 AND MALT1 | Klemm et al. ARTICLE We addressed the immediate and the late phase PCA re- sponses separately (Fig. 2). For early phase PCA reactions, mice were primed by in- tradermal injection of monoclonal anti-dinitrophenyl (anti- DNP) IgE antibody into the ear. 24 h later, the animals were i.v. coinjected with Evans blue dye and the antigen DNP coupled to HSA (DNP–HSA). The extravasation of Evans blue dye during the fi rst hour of the PCA reaction is mainly dependent on the degranulation of activated mast cells with rapid histamine and serotonin release resulting in locally in- creased blood vessel permeability (21). The Evans blue dye extravasation was monitored by inspection and quantifi ed 60 min after antigen challenge (Fig. 2, A and B). Neither the extravasation kinetics nor the total amount of Evans blue dye in the ear were substantially diff erent in the three genotypes, indicating that the mast cell–mediated immediate phase PCA reaction does not require Bcl10 or Malt1. We next examined the late phase PCA response that is promoted by mast cell–derived proinfl ammatory cytokines (1, 23, 24). Mice were sensitized i.v. with anti-DNP IgE an- tibody. 24 h later, the hapten dinitrofl uorobenzene (DNFB; 0.2% wt/vol) was epicutaneously applied to both sides of the ears. Although WT mice exhibited the anticipated promi- nent edema as early as 6 h and reached a maximum 24 h after −/− −/− antigen stimulation, both Bcl10 and Malt1 mice showed only a minor response (Fig. 2 C). Even 24 and 48 h after antigen challenge, the increase in ear thickness in the mutant animals reached <20% of that observed in WT mice. Therefore, we conclude that both Bcl10 and Malt1 are es- sentially required for regular IgE-dependent late phase PCA reactions in vivo. Figure 1. Mast cell development in the absence of Bcl10 or Malt1. Bcl10 and Malt1 differentially regulate Fc𝛆 RI-mediated −/− −/− (A and B) Skin biopsies from WT, Bcl10 , and Malt1 mice. (A) Repre- degranulation, lipid mediator secretion, sentative areas from the ear skin show the presence of toluidine blue– and cytokine production positive mast cells in all three genotypes (arrowheads) at 20×. Insets at To provide a basis for the defective PCA reactions in Bcl10- 100× show the boxed mast cells from each section. Bars: (full images) 10 μm; (insets) 2 μm. (B) Mast cell frequencies in skin sections from the and Malt1-defi cient mice, we examined the capacities of −/− −/− ear, neck, and groin (n = 3 mice/genotype). There was no important dif- Bcl10 , Malt1 , and WT mast cells to degranulate, syn- ference among the three genotypes. Data are means ± SEM. (C) Flow thesize, and secrete lipid mediators and to produce cytokines −/− −/− cytometric analysis of BMMCs from WT, Bcl10 , and Malt1 mice. (Fig. 3 and see Fig. 4). These and all subsequent analyses were Percentages of FcεRI and c-kit double-positive mast cells are indicated. performed on BMMCs in vitro. Results are representative of fi ve different experiments. (D) Bcl10 and −/− −/− WT, Bcl10 , and Malt1 BMMCs were loaded with −/− Malt1 expression levels in BMMCs. Western blots of WT, Bcl10 , and −/− antigen-specifi c IgE and subsequently activated by FcεRI Malt1 BMMCs with antibodies against Bcl10, Malt1, and β-actin. cross-linking with increasing doses of multivalent antigen to Results are representative of fi ve independent experiments. induce cell degranulation (Fig. 3 A). Mast cells release the enzyme β-hexosaminidase from intracellular granules whose −/− Malt1 mast cells and the Malt1 protein concentration is activity in the culture supernatant can be quantifi ed to deter- −/− smaller in Bcl10 BMMCs compared with the WT, sug- mine the effi ciency of mast cell degranulation (25). Consis- gesting that cellular levels of each of the two binding partners tent with the normal Evans blue dye extravasation during the depend on the presence of the other. immediate phase PCA reaction in vivo, mast cells derived from Bcl10- or Malt1-defi cient mice released similar amounts of β-hexosaminidase as WT BMMCs in response to FcεRI Bcl10 and Malt1 are required for normal mast cell function in vivo ligation. We also stimulated cells with PMA together with To test whether Bcl10 or Malt1 play a functional role in mast the calcium ionophore ionomycin (Iono). These pharmaco- cells in vivo, we performed two types of FcεRI-mediated logical agents bypass FcεRI-proximal signaling events and passive cutaneous anaphylaxis (PCA) experiments (21, 22). stimulate cells directly by mobilizing free calcium ions and JEM VOL. 203, February 20, 2006 339 Figure 2. Normal immediate but defective late phase PCA in statistically signifi cant ( P ≥ 0.05). (C) Late phase PCA reactions. WT, −/− −/− −/− −/− Bcl10 and Malt1 mice. (A and B) Immediate phase PCA reactions. Bcl10 , and Malt1 mice were passively sensitized by i.v. injection of Mice were passively sensitized by intradermal injection of anti-DNP IgE anti-DNP IgE. 24 h later, they were challenged by epicuta-neous applica- into the left ear. The right ear was injected with PBS. Subsequently, mice tion of DNFB to both ears, and ear thickness was measured over time. were challenged by i.v. injection of antigen in PBS/Evans blue. (A) Ears The increase in ear thickness was calculated as described in Material and −/− −/− from WT, Bcl10 , and Malt1 mice 1 h after antigen challenge. (B) The methods. Results are representative of three independent experiments. IgE-mediated Evans blue extravasation was calculated as the difference in Data are presented as means ± SEM. * and **, P ≤ 0.05 and P ≤ 0.02 −/− −/− the amount of extravasated dye in the IgE-sensitized and the nonsensi- between WT and Bcl10 or WT and Malt mice, respectively. tized control ears. Differences between the three geno-types were not −/− −/− activating PKC enzymes. Again, no substantial diff erences in of stimulation. In sharp contrast, Bcl10 and Malt1 β-hexosaminidase activity were detected in the supernatants BMMCs produced only minute amounts of TNF-α or IL-6 of BMMCs of the three genotypes (Fig. 3 A). even at later time points. To further characterize the defects Furthermore, we investigated the production of proin- in TNF-α and IL-6 production in Bcl10- and Malt1-defi - −/− −/− fl ammatory lipid mediators in WT, Bcl10 , and Malt1 cient BMMCs, we analyzed cytokine mRNA levels before BMMCs in response to FcεRI ligation using an enzyme- and 30 min after FcεRI ligation using semiquantitative RT- linked immunoassay (Fig. 3 B). Mast cells of the three geno- PCR. Unstimulated and stimulated populations of all three types produced equal amounts of the leukotrienes C , D , cell lines contained equal amounts of β-actin transcripts, indi- 4 4 and E (LTC , LTD , and LTE ). BMMC activation with cating appropriate normalization (Fig. 4 B). In WT BMMCs, 4 4 4 4 PMA/Iono also induces robust leukotriene production. both TNF-α and IL-6 mRNA levels increased substantially −/− Again, no diff erences were found between WT, Bcl10 , upon FcεRI stimulation. However, in the absence of Bcl10 −/− and Malt1 BMMCs (Fig. 3 B). Collectively, these fi rst sets or Malt1 the induction of these cytokine transcripts was ei- of in vitro experiments show that the signaling proteins Bcl10 ther absent or largely reduced compared with the WT. Thus, and Malt1 are dispensable for mast cell degranulation or leu- Bcl10 and Malt1 are critically required for FcεRI-mediated kotriene synthesis in response to FcεRI ligation or PMA/ TNF-α and IL-6 gene transcription and subsequent protein Iono treatment. production. These results indicate that the defective PCA re- It is known that the late phase PCA response in vivo is at actions observed in Bcl10- or Malt1-defi cient mice in vivo least in part induced by proinfl ammatory cytokines, in partic- might refl ect the essential requirements for the two signaling ular TNF-α, released from activated mast cells (1, 23, 24). proteins for FcεRI-mediated cytokine production. Therefore, we were especially interested in measuring the transcriptional induction, production, and release of cyto- Bcl10 and Malt1 are dispensable for IgE-induced mast −/− −/− kines by Bcl10 and Malt1 mast cells in response to cell survival FcεRI stimulation in vitro. To this end, we fi rst performed In lymphocytes, Bcl10 and Malt1 are not only vital for cell time course experiments and analyzed the concentrations of activation but also involved in the regulation of cell survival TNF-α and IL-6 in the supernatant of stimulated cells by (15). Because recent studies demonstrated a role for FcεRI- ELISA. WT mast cells produced both mediators readily (Fig. derived signals in the control of mast cell survival (4, 5), we 4 A). The maximal concentrations were detected after 2 h also studied the contributions of Bcl10 and Malt1 to this 340 REGULATION OF FCεRI SIGNALING BY BCL10 AND MALT1 | Klemm et al. ARTICLE Figure 3. Normal degranulation and leukotriene synthesis in −/− −/− −/− Bcl10 and Malt1 BMMCs. (A) Regular degranulation of Bcl10 −/− −/− −/− and Malt1 mast cells. BMMCs from WT, Bcl10 , and Malt mice were loaded with anti-DNP IgE and subsequently stimulated for 30 min with the indicated concentrations of DNP–HSA. Alternatively, BMMCs were left unsensitized and stimulated with PMA/Iono (100 nM each) for 30 min. The extent of degranulation was determined by measuring the activity of the granular enzyme β-hexosaminidase in supernatants and cell lysates and calculated as described in Material and methods. Data are means ± SEM from triplicate samples and are representative of four in- −/− dependent experiments. (B) Regular leukotriene synthesis in Bcl10 and −/− Malt1 mast cells. Anti-DNP IgE-sensitized BMMCs from all three geno- types were stimulated with DNP–HSA at the indicated concentrations, or −/− −/− Figure 4. Defective cytokine production in Bcl10 and Malt1 BMMCs were left unsensitized and stimulated with PMA/Iono (100 nM −/− −/− BMMCs. (A) Impaired cytokine secretion in Bcl10 and Malt1 mast each). 60 min later, the supernatants of the stimulated cells were col- −/− −/− cells. WT, Bcl10 , and Malt1 BMMCs were loaded with anti-DNP IgE lected and assayed for the concentrations of LTC , LTD , and LTE . Data 4 4 4 and stimulated with DNP–HSA for the indicated intervals. Supernatants are means ± SEM from triplicate samples and representative of four were collected, and TNF-α and IL-6 protein concentrations were deter- independent experiments. mined by ELISA. Data are means ± SEM from triplicate samples and rep- resentative of at least fi ve independent experiments. * and **, P ≤ 0.05 −/− −/− and P ≤ 0.01 between WT and Bcl10 or WT and Malt1 BMMCs, −/− −/− −/− pathway (Fig. 5). BMMCs of WT, Bcl10 , and Malt1 respectively. (B) Defective expression of cytokine mRNAs in Bcl10 and −/− −/− −/− Malt1 mast cells. BMMCs from WT, Bcl10 , and Malt1 mice were mice were incubated for 4 d in media with low concentra- stimulated for 30 min as in A. RNA was purifi ed, and TNF-α, IL-6, and tions of FCS without IL-3 and SCF. This growth factor −/− β-actin mRNAs were detected by semiquantitative RT-PCR using specifi c withdrawal induced apoptotic death of WT, Bcl10 , and primers. Data are representative of three independent experiments. −/− Malt1 BMMCs. The frequency of apoptotic cells was in- dependent of the presence of Bcl10 or Malt1 as determined by fl ow cytometry after annexin V/propidium iodide stain- ing. (Fig. 5 A). Stimulation of cells with IgE alone or in com- defi cient mast cells, we analyzed the signaling pathways bination with antigen (unpublished data) rescued cell death downstream of FcεRI stimulation (Fig. 6 and see Fig. 7). and up-regulated the expression of the prosurvival protein Ligation of IgE-occupied FcεRI molecules with antigen Bcl-X in mast cells of all three genotypes similarly (Fig. 5 B). results in the rapid activation of receptor-proximal tyrosine We thus conclude that Bcl10 and Malt1 are not essential for kinases, which is a prerequisite for all mast cell eff ector func- the FcεRI-controlled survival pathway. tions (2). Consistent with the normal degranulation and lipid −/− −/− mediator synthesis in Bcl10 and Malt1 mast cells, re- Bcl10 and Malt1 are essential regulators ceptor-proximal signaling does not involve Bcl10 or Malt1 of Fc𝛆 RI-controlled NF-𝛋 B activation because the tyrosine phosphorylation patterns induced by To defi ne the molecular mechanisms responsible for the FcεRI stimulation did not diff er in kinetics or intensity defective cytokine gene expression in Bcl10- and Malt1- among BMMCs of the three genotypes (Fig. 6 A). JEM VOL. 203, February 20, 2006 341 mediated NF-κB activation. To test this hypothesis, we analyzed IκB-α phosphorylation and its subsequent degra- dation in FcεRI-stimulated BMMCs of the three geno- types. FcεRI signaling in WT cells induced rapid IκB-α phosphorylation and, consequently, its degradation (Fig. 7 A). In contrast, signaling to IκB-α was completely abol- −/− −/− ished in Bcl10 or in Malt1 mast cells. Direct PKC activation via PMA/Iono stimulation also resulted in IκB-α phosphorylation and degradation only in the WT cells but not in BMMCs defi cient for either Bcl10 or Malt1, indi- cating a requirement for Bcl10 and Malt1 downstream of PKC activation. To directly investigate NF-κB nuclear translocation and DNA binding, we performed gel shift assays with nuclear ex- tracts from stimulated mast cells. Consistent with the marked impairment of IκB-α phosphorylation and degradation in −/− −/− Bcl10 and Malt1 BMMCs, NF-κB was only activated in WT mast cells but not in those defi cient for Bcl10 or Malt1 (Fig. 7 B). Thus, Bcl10 and Malt1 are both essential to spe- cifi cally couple FcεRI and PKC signaling to the activation of the NF-κB pathway in mast cells. Figure 5. Regular IgE-induced survival signaling in the absence of Bcl10 or Malt1. (A) BMMCs from all three genotypes were deprived of DISCUSSION the mast cell survival cytokines IL-3 and SCF and cultured in media with In this paper, we have demonstrated on a genetic basis that low concentrations of FCS (1%) with or without 5 μg/ml anti-DNP IgE Bcl10 and Malt1 are essential and nonredundant positive reg- mAb. 48 and 96 h later, apoptotic cell death was determined by fl ow ulators of mast cell activation and eff ector function. Although cytometry after annexin V/propidium iodide staining. The percentages of mast cells seem to diff erentiate and survive normally in the viable mast cells are shown. Data are means ± SEM from triplicate sam- ples and are representative of four independent experiments. No impor- absence of either Bcl10 or Malt1, both proteins are critically tant differences were detected among the three genotypes. (B) BMMCs required for NF-κB activation induced by the high-affi nity were cultured as in A and left unstimulated or stimulated for 24 h with IgE receptor FcεRI. Bcl10- or Malt1-defi cient mast cells do 5 μg/ml anti-DNP IgE. Total cell lysates were resolved by SDS-PAGE and not produce the NF-κB–regulated proinfl ammatory cyto- immunoblotted with anti–Bcl-X antibody and, after stripping, reprobed kines TNF-α and IL-6 and, consistently, late phase PCA re- with anti–β-actin antibody to control for equal loading. actions are severely impaired in Bcl10- or Malt1-defi cient mice in vivo. Activation of mast cells through the FcεRI initiates a FcεRI-proximal signaling activates the Erk, Jnk, and cascade of events that lead to degranulation, production of p38 pathways, as well as the phosphatidylinositol 3–kinase lipid mediators such as leukotrienes, and transcriptional up- pathway that results in the activation of the serine kinase regulation and release of proinfl ammatory cytokines such as Akt. These cascades regulate both cytokine production and TNF-α or IL-6. These all represent normal mast cell func- generation of arachidonic acid metabolites upon FcεRI li- tions that, depending on the context, may be benefi cial or gation (1, 2). Bcl10 and Malt1 have been implicated in the harmful in the setting of innate or allergic immune responses. regulation of Jnk and p38 signaling in lymphocytes (18, 19). Bcl10 and Malt1 are not involved in FcεRI-proximal signal- However, using immunoblotting with activation state–spe- ing events that are known to lead to the activation of PKC cifi c phosphoantibodies against Jnk, p38, the p42 and p44 enzymes (2). The PKC family consists of at least 10 serine/ MAP kinases Erk1 and Erk2, or Akt, we could not detect threonine kinases, and previous studies have indicated that diff erences in the FcεRI-induced activation of any of these PKC activation is involved in all aspects of mast cell eff ector −/− −/− pathways among WT, Bcl10 , and Malt1 mast cells functions (26). Although comprehensive studies of the indi- (Fig. 6 B). We additionally activated the cells with PMA/ vidual roles of all PKC isoforms in mast cell biology are Iono and again detected similar activation for Jnk, p38, missing, chemical or genetic inhibition of the PMA- and cal- Erk1, Erk2, and Akt in the presence or absence of either cium-sensitive conventional isoforms PKCα and PKCβ Bcl10 or Malt1 (Fig. 6 C). identifi ed these kinases as essential inducers of degranulation As earlier mentioned, the expression of TNF-α and IL- and cytokine and leukotriene production (27–29). The 6 critically depends on the activation of NF-κB. Based on PMA-sensitive but calcium-insensitive isoform PKCδ is in- known functions of Bcl10 and Malt1 in lymphocytes and volved in the activation of leukotriene synthesis (30) and can their newly discovered requirement in mast cells, we rea- simultaneously function as a negative regulator of antigen- soned that the two proteins might be involved in FcεRI- induced degranulation (31). 342 REGULATION OF FCεRI SIGNALING BY BCL10 AND MALT1 | Klemm et al. ARTICLE In this paper, we show that FcεRI- and PMA/Iono-in- duced IκB-α phosphorylation, degradation, and NF-κB ac- tivation are completely abolished in Bcl10- or Malt1-defi cient mast cells. These results reveal an essential requirement for Bcl10–Malt1 signaling downstream of the FcεRI and down- stream of all PMA-responsive PKCs in mast cells. Impor- tantly, however, FcεRI- or PMA/Iono-induced activation −/− of the Jnk, p38, Erk, and Akt pathways are intact in Bcl10 −/− and Malt1 mast cells, indicating that the Bcl10–Malt1 complex does not aff ect all PKC downstream pathways. Consistent with the regular activation of selected FcεRI or PKC eff ector signals, we observed normal mast cell survival, degranulation, and leukotriene production in the absence of Bcl10 or Malt1. The observation that TNF-α and IL-6 transcription and protein production are severely impaired in Bcl10- or Malt1- defi cient mast cells is in agreement with our fi ndings that Bcl10–Malt1 specifi cally controls FcεRI- induced NF-κB activation and the known role of NF-κB for cytokine gene expression. Considering all in vitro results collectively, we propose a model for the molecular func- tions of Bcl10 and Malt1 in mast cell signaling that is de- picted in Fig. 7 C. This signaling model is consistent with the in vivo observations in Bcl10- and Malt1-defi cient mice, as immediate phase PCA reactions that depend on mast cell degranulation (21) are normal, whereas late phase reactions that are at least partially dependent on transcriptional induc- tion and the release of mast cell–derived cytokines (1, 24) are severely impaired in the absence of either Bcl10 or Malt1. However, because in the mutant animals all cells lack Bcl10 or Malt1, signaling defi ciencies in other cells besides mast cells could additionally contribute to the defective late phase PCA response. Experiments with genetically altered mice have recently elucidated many aspects of positive and negative regulation of FcεRI-induced mast cell activation (1). Collectively, these fi ndings demonstrate that antigen receptor–specifi c mole- cules required for FcεRI signaling generally aff ect several as- pects of mast cell function simultaneously. In contrast, we identifi ed the Bcl10–Malt1 signaling complex as a molecular structure that selectively regulates one downstream eff ector arm of FcεRI, namely NF-κB–mediated cytokine produc- tion, but does not aff ect mast cell survival, degranulation, or the synthesis of leukotrienes. The conclusion that the Bcl10–Malt1 complex operates selectively downstream of PKCs in FcεRI signaling to NF-κB are shown on the left. Data are representative of three independent ex- periments. (B) Immunoblot with anti–phospho-p38, anti–phospho- Figure 6. Normal Fc𝛆 RI proximal signaling and regular p38, Erk, p44/42, anti–phospho-Jnk, and anti–phospho-Akt. After stripping, −/− −/− Jnk, and Akt activation in Bcl10 and Malt1 mast cells. membranes were reprobed with anti-p38, anti-p44/p42, anti-Jnk, and −/− −/− (A and B) WT, Bcl10 , and Malt1 BMMCs sensitized with anti-DNP anti-Akt antibody, respectively. Data are representative of at least three −/− −/− IgE were stimulated with DNP–HSA for the indicated time intervals (in independent experiments. (C) Unsensitized WT, Bcl10 , and Malt1 minutes) as described in Material and methods. Total cell lysates were BMMCs were stimulated with PMA/Iono for the indicated time intervals separated by SDS-PAGE (2 × 10 cell equivalents). (A) Immunoblot (in minutes) as described in Material and methods and immunoblotted as with anti-phosphotyrosine antibodies. Molecular mass (kD) markers in B. Results are representative of three independent experiments. JEM VOL. 203, February 20, 2006 343 Figure 7. Bcl10 and Malt1 are required for NF-𝛋 B signaling in PMA/Iono (100 nM each) for 60 min. Subsequently, nuclear extracts were mast cells. (A) Defective IκB-α phosphorylation and degradation after prepared and subjected to gel mobility shift assays using a radiolabeled −/− −/− FcεRI ligation or PMA/Iono treatment in Bcl10 or Malt1 mast cells. probe containing NF-κB binding site sequences. Results are representa- BMMCs from all genotypes were sensitized with anti-DNP IgE and stimu- tive of three independent experiments. (C) A model for the role of Bcl10 lated with DNP–HSA for the indicated time intervals (in minutes). Alterna- and Malt1 in FcεRI-mediated signal transduction after FcεRI receptor tively, cells were left unsensitized and stimulated with PMA/Iono (100 nM stimulation, Bcl10 and Malt1 operate downstream of PKCs and serve each). IκB-α phosphorylation and degradation were determined by West- as selective signal transducers that activate the IκB-dependent NF-κB ern blotting. Membranes were reprobed with anti–β-actin antibody to pathway. The Bcl10–Malt1 complex selectively controls proinfl ammatory control for equal loading. Data are representative of at least four inde- cytokine production but does not regulate mast cell degranulation or −/− pendent experiments. (B) Defective NF-κB activation in Bcl10 and leukotriene synthesis. For details, see Discussion. −/− Malt1 mast cells. BMMCs were left unstimulated or stimulated with induced NF-κB activation but selectively controls p38 MAP provides novel insights into context-specifi c immune regu- lation. The FcεRI on mast cells belongs to the family of kinase signaling in response to BCR ligation (40). Our fi nd- multisubunit immunoreceptors that also includes the TCR ings that FcεRI signaling requires both Bcl10 and Malt1 for and the B cell receptor (BCR), but numerous genetic stud- NF-κB control but not for Jnk or p38 activation extend this ies have demonstrated that each of these receptors uses idea of receptor-specifi c utilization of the signaling module unique sets of downstream molecules to diff erentially medi- and reinforces the necessity to investigate the precise func- ate cell activation. For example, the FcεRI requires the ty- tion of each signal transducer in distinct cell lineages. So far rosine kinases Syk and Btk and the adaptor molecule linker there are no other reports available that explore roles of for activation of T cells to mediate mast cell activation (32– Bcl10, Malt1, or any of their binding partners in FcεRI sig- 34). Syk and Btk are additionally required for BCR signal- naling. It will thus be additionally important to study mast cells in mice defi cient in Carma proteins, TNF receptor– ing but are completely dispensable for T cell activation, whereas the linker for activation of T cells is essential for associated factor molecules, and MAP kinase–activating ki- TCR signaling but has no role in B cells (35–37). The nases, including TAK1, to understand precisely how the Bcl10–Malt1 complex is also known to be diff erentially used FcεRI diff erentially couples to NF-κB and Jnk/p38 down- by immunoreceptors. Bcl10 can bind to Carma molecules stream signaling. These studies could also reveal the Bcl10– and recruit Malt1 and various TNF receptor–associated fac- Malt1–independent mechanisms of Jnk and p38 activation tor molecules and kinases such as TAK1 into signaling com- and give further insights into the selective control of distinct plexes to mediate downstream activation of NF-κB, Jnk, mast cell eff ector functions. and p38 (15, 19, 38–40). TCR signaling to NF-κB critically Recent biochemical studies have shown that Bcl10–Malt1 depends on Bcl10, Malt1, and the kinase TAK1 (39). How- signaling to IKK and NF-κB involves the oligomerization of Bcl10 and Malt1 and subsequent Malt1-dependent regu- ever, Malt1 is partially expendable for BCR signaling to NF-κB (18), and TAK1 is completely dispensable for BCR- latory lysine-63–linked ubiquitinylation of IKKγ/NEMO, 344 REGULATION OF FCεRI SIGNALING BY BCL10 AND MALT1 | Klemm et al. ARTICLE Mast cell degranulation. To induce degranulation, 2 × 10 BMMCs/ml which induces IKK activity (39, 41). These fi ndings open were loaded with 5 μg/ml anti-DNP IgE mAb (SPE-7; Sigma-Aldrich) for the possibility that Bcl10–Malt1 signaling could potentially 1 h on ice in Tyrode’s buff er (10 mM Hepes, pH 7.4, 130 mM NaCl, 5 mM be inhibited by targeted drugs because numerous kinase KCl, 1.4 mM CaCl , 1 mM MgCl , 5.6 mM glucose, and 0.1% BSA). After 2 2 inhibitors and many compounds that target the ubiquitin/ washing, sensitized cells were stimulated with the concentrations of DNP– proteasome system are in preclinical or clinical development. HSA indicated in the fi gures. Alternatively, cells were left unsensitized and Mast cell–derived TNF-α recruits and activates neutrophils stimulated with 100 nM PMA (Sigma-Aldrich) and 100 nM Iono (Sigma- Aldrich) in Tyrode’s buff er. The enzymatic activities of β-hexosaminidase in and lymphocytes at sites of infl ammation and has been impli- supernatants and cell pellets solubilized with 0.5% Triton X-100 in Tyrode’s cated in tissue remodeling processes, angiogenesis, and fi bro- Buff er were measured with p-nitrophenyl-N-acetyl-β-d-glucosaminide sis that can be so prominent in IgE-associated chronic allergic (Sigma-Aldrich), and the percentage of degranulation was calculated as pre- diseases (1, 23, 42, 43). Consequently, there is a tremendous viously described (25). interest in inhibiting TNF-α production in allergic infl am- mation. In this paper, we demonstrate that a complete ge- 6 Measurements of leukotrienes and cytokines. 2 × 10 BMMCs/ml netic blockage of Bcl10–Malt1 signaling thoroughly abolishes were loaded with 0.5 μg/ml anti-DNP IgE mAb (SPE-7) overnight, washed twice, and stimulated with DNP–HSA or left unsensitized and stimulated FcεRI-specifi c NF-κB activation and TNF-α and IL-6 pro- with PMA and Iono (100 nM each) as indicated in the fi gures. Cell superna- duction in mast cells. Thus, we suggest that specifi c thera- tants were harvested, and leukotrienes LTC , LTD , and LTE were deter- 4 4 4 peutic targeting of Bcl10–Malt1 signaling could potentially mined by enzyme-linked immunoassay (GE Healthcare), whereas IL-6 and contribute to the therapy of mast cell–mediated infl amma- TNF-α were determined by ELISA DuoSets (R&D Systems), as recom- tory diseases without the toxic side eff ects that can be ex- mended by the manufacturer. pected from general NF-κB inhibition. RT-PCR. 2 × 10 BMMCs/ml were loaded with 0.5 μg/ml anti-DNP IgE mAb (SPE-7) overnight, washed twice, and stimulated with 20 ng/ml MATERIALS AND METHODS DNP–HSA for 30 min. Total RNA was isolated using TRIzol reagent Mice. The generation of Bcl10- and Malt1-defi cient mice was reported (Invitrogen), according to the manufacturer’s instructions, and reverse tran- previously (17, 18). Mice were housed under specifi c pathogen-free condi- scribed using the Superscript fi rst-strand synthesis system (Invitrogen). Prim- tions. The study was conducted under federal guidelines for the use and care ers and PCR conditions were described elsewhere (25). of laboratory animals and was approved by the Government of the District of Upper Bavaria. Mice were of a C57BL/6 and 129/J mixed background. Survival assay. BMMCs were cultured as indicated in the fi gures. Cell via- Littermates were used in each experiment. bility was quantifi ed by fl ow cytometry after annexin V/propidium iodide staining as recommended by the manufacturer (BD Biosciences). Skin histology. Naive animals at 10–12 wk of age were killed, and skin samples from the ear, neck, and groin regions were removed and fi xed in 4% Signal transduction. For FcεRI signaling, 2 × 10 BMMCs/ml were formalin. 4-μm tissue sections were stained with toluidine blue, and mast preloaded with 0.5 μg/ml anti-DNP IgE mAb (SPE-7) and subsequently cells were counted in the dermis of each sample at 40×. 10 fi elds at 10× (per activated by adding 20 ng/ml DNP–HSA, as indicated in the fi gures. Alter- mouse and location) were used to delineate and integrate the dermal area natively, BMMCs were left unsensitized and stimulated with PMA and (KS 300 software; Carl Zeiss MicroImaging, Inc.) that was analyzed. Iono (100 nM each) or with 5 μg/ml anti-DNP IgE mAb for Bcl-X in- duction. Cells were lysed, and denatured proteins were separated on 10% BMMC culture and FACS analysis. Bone marrow cells were cultured in polyacrylamide gels and subjected to immunoblotting using antibodies suspension in RPMI 1640 with 20% FCS, 30 ng/ml murine IL-3 (R&D against phosphotyrosine (PY99; Santa Cruz Biotechnology, Inc.), phos- Systems), 50 ng/ml murine SCF (R&D Systems), and 150 μM monothio- pho-p38, p38, phospho-p44/42, p44/42, phospho-Jnk, Jnk, phospho-Akt, glycerol (Sigma-Aldrich). To quantify BMMC frequencies, cells were stained Akt, phospho–IκB-α, Iκb-α, Bcl-X (all from Cell Signaling), or β-actin with FITC-labeled anti-FcεRI and PE-labeled anti–c-kit antibody (eBiosci- (Sigma-Aldrich). For electromobility shift assays, 10 BMMCs/sample were ence) and analyzed by fl ow cytometry (FACScan; Becton Dickinson). stimulated with PMA/Iono, and gel shifts were performed as described previously (17). PCA. For immediate phase PCA reactions, mice were passively sensitized by intradermal injection of 250 ng anti-DNP IgE mAb (clone H1-ε-26; Statistical analysis. Results were analyzed for statistical signifi cance with provided by F.-T. Liu [University of California, Davis, Sacramento, CA] the unpaired two-tailed Student’s t test (Excel; Microsoft). Data from and D.H. Katz [Avanir Pharmaceuticals, San Diego, CA]) into one ear and −/− −/− Bcl10 and Malt1 mice or BMMCs were separately compared with the PBS injection into the contralateral ear. 24 h later, mice were challenged by WT. Diff erences between groups were considered signifi cant at P ≤ 0.05. i.v. injection of 150 μg of DNP–HSA (30–40 moles DNP/mol HSA; Sigma-Aldrich) in 100 μl Evans blue dye (0.5%; Sigma-Aldrich). To deter- We thank Michael Huber and Margot Thome for helpful discussions and advice; mine the amount of extravasated dye, mice were killed, ears were removed, Frank Redegeld for affi nity purifying H1-ε-s26 mAb; and Britta Dorn for excellent and Evans blue dye was extracted with potassium hydroxide and photomet- technical assistance. rically quantifi ed as described previously (22). This work was supported by grants from the German National Genome Project For late phase PCA reactions, mice were passively sensitized by i.v. (NUWS31T05S15T03, subproject 3b), the German Federal Ministry of Science and injection of 2 μg anti-DNP IgE mAb (clone H1-ε-26) or left unsensitized. Education (BMBF 01GC0104, subproject 2), and the Technical University of Munich 24 h later, sensitized and nonsensitized mice were challenged by epicutane- Medical School (KKF 8760179) to T. Jakob and by a Max-Eder-Program grant from ous application of 10 μl of DNFB (0.2% wt/vol) in acetone/olive oil (4:1) Deutsche Krebshilfe and grants from the Deutsche Forschungsgemeinschaft (SFB to both sides of both ears. The ear swelling response was assessed by measur- 455 and SFB 456) to J. Ruland. ing the ear thickness using an engineer’s micrometer dial thickness gauge. The authors have no confl icting fi nancial interests. The increment of ear thickness (postchallenge value – prechallenge baseline value) was expressed as the percentage of the baseline value obtained before Submitted: 4 October 2005 antigen challenge. Accepted: 23 December 2005 JEM VOL. 203, February 20, 2006 345 21. Inagaki, N., S. Goto, H. Nagai, and A. Koda. 1986. Homologous pas- REFERENCES sive cutaneous anaphylaxis in various strains of mice. Int. Arch. Allergy 1. Galli, S.J., J. Kalesnikoff , M.A. Grimbaldeston, A.M. Piliponsky, C.M. Williams, and M. Tsai. 2005. Mast cells as “tunable” eff ector and Appl. 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The Journal of Experimental Medicine – Pubmed Central

Published: Feb 20, 2006

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