Cellular Signalling 21 (2009) 79–86
`
`Contents lists available at ScienceDirect
`
`Cellular Signalling
`
`Cellular
`Signalling
`- •-=:.;;:::_.
`
`.....LS~VIER
`
`j o u r n a l h om e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g
`
`Bruton's tyrosine kinase is dispensable for the Toll-like receptor-mediated activation
`of mast cells
`Carolin N. Zorn a, Simone Keck a, Rudi W. Hendriks b, Michael Leitges c,
`Marina A. Freudenberg a, Michael Huber a,d,⁎
`a Department of Molecular Immunology, Biology III, Albert-Ludwigs-University Freiburg and Max-Planck-Institute for Immunobiology, 79108 Freiburg, Germany
`b Department of Pulmonary Medicine, Erasmus Medical Center Rotterdam, NL-3000 CA Rotterdam, The Netherlands
`c Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway
`d Centre for Biological Signaling Studies (bioss), Albert-Ludwigs-University Freiburg, Germany
`
`a r t i c l e
`
`i n f o
`
`a b s t r a c t
`
`Article history:
`Received 21 May 2008
`Received in revised form 11 September 2008
`Accepted 22 September 2008
`Available online 26 September 2008
`
`Keywords:
`Bruton's tyrosine kinase
`Mast cells
`Macrophages
`Lipopolysaccharide
`Lipopeptide
`Protein kinase C-β
`Signal transduction
`
`1. Introduction
`
`Bruton's tyrosine kinase (Btk) represents an important signaling element downstream of ITAM-containing
`receptors, e.g. FcεR1 and BCR. Btk is part of the calcium signalosome and thus, critically involved in
`intracellular calcium mobilization. Loss of Btk or expression of mutant forms results in severe disease
`phenotypes, X-linked agammaglobulinemia (XLA) and Xid in humans and mice, respectively. Previously,
`roles for Btk in TLR-mediated signal transduction have been found in monocytes/macrophages. In the present
`study we show that Btk deficiency moderately enhances or has no influence on the LPS- or lipopeptide-
`induced secretion of IL-6 and TNF-α from murine bone marrow-derived mast cells (BMMCs).
`Furthermore, activation of p38 kinase, which is required for cytokine production, is comparable in WT and
`Btk−/− BMMCs. Moreover, stability of the adaptor protein Mal as well as LPS-induced H2O2 production does
`not vary between WT and Btk−/− cells. Interestingly, PKC-β deficiency, which results in a Xid-like phenotype
`as well, has also no negative effect on LPS-induced cytokine secretion, suggesting that proteins of the calcium
`signalosome are not involved in TLR-mediated BMMC activation. In conclusion, the study reveals that Btk is
`dispensable for TLR signaling and function in murine BMMCs.
`
`© 2008 Elsevier Inc. All rights reserved.
`
`Btk is a cytoplasmic tyrosine kinase expressed in certain
`hemopoietic cells including B-lymphocytes, macrophages, mast
`cells, and platelets [1]. The importance of Btk is particularly obvious
`during B cell development and activation [2,3]. Btk deficiency
`manifests as X-linked agammaglobulinemia (XLA) and Xid in humans
`and mice, respectively [1]. Not only complete absence of Btk, but also
`defects in Btk, like point mutations in the PH and SH3 domains, can
`cause this severe disease [4]. Due to its role in B cell development, Btk-
`deficient patients/mice lack mature B cells and express dramatically
`reduced amounts of immunoglobulins [2]. Btk is part of the so-called
`calcium signalosome downstream of the BCR. It participates in the
`
`Abbreviations: BMMC, bone marrow-derived mast cell; BMMΦ, bone marrow-
`derived macrophage; Btk, Bruton's tyrosine kinase; LP,
`lipopeptide; Mal, MyD88
`adapter-like protein; MC, mast cell; SOCS1, suppressor of cytokine signaling 1; XLA, X-
`linked agammaglobulinemia.
`⁎ Corresponding author. Molecular Immunology, Institute for Biology III, University of
`Freiburg, Germany and Max-Planck-Institute for Immunobiology Stübeweg 51, 79108
`Freiburg, Germany. Tel.: +49 761 5108 438; fax: +49 761 5108 423.
`E-mail address: huberm@immunbio.mpg.de (M. Huber).
`
`0898-6568/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
`doi:10.1016/j.cellsig.2008.09.010
`
`activation of PLC-γ2 and thus regulates the release of intracellular
`calcium from the endoplasmic reticulum [3]. The same function for
`Btk has been reported in mast cells (MCs) activated via the FcεR1 [5].
`Both FcεR1 and BCR are ITAM-containing receptors [6] and share
`elements and mechanisms of signal transduction [5].
`Btk has also been reported to be involved in the signal transduction of
`ITAM-independent receptor systems, e.g. TLR. However, the current
`literature provides in part conflicting data. Btk has been found to interact
`with components of the TLR4 and TLR2 signaling machinery, i.e. the
`adapter proteins, MyD88 and MyD88 adapter-like protein (Mal), as well
`as the interleukin-1 receptor-associated kinase-1 in HEK293 transfec-
`tants [7], and to be involved in transactivation of NFκB [8]. Furthermore,
`it was reported that Mal is subject to Btk-mediated tyrosine phosphor-
`ylation in response to TLR4 or TLR2 engagement [9] and this
`phosphotyrosine serves as an acceptor site for the suppressor of
`cytokine signaling 1 (SOCS1) protein. Binding of SOCS1 to Mal conveys
`this adaptor protein for degradation by the 26S proteasome [10]. Thus,
`Btk seems to be acting as part of a negative regulatory mechanism
`limiting primary innate immune responses. In accordance, degradation
`of Mal was absent in LPS-stimulated mouse Xid splenocytes [10]. From
`the involvement of Btk in degradation of Mal one would expect that cells
`lacking Btk function exhibit enhanced reactivity to LPS. However, the
`
`

`

`80
`
`C.N. Zorn et al. / Cellular Signalling 21 (2009) 79–86
`
`opposite effect of Btk deficiency, an impaired TNF-α response to LPS, was
`observed in mononuclear cells from XLA patients [11]. XLA mononuclear
`cells showed attenuated LPS-induced phosphorylation of p38 kinase
`compared to cells from control donors and this seemed to cause
`decreased stability of TNF-α mRNA in XLA cells [11]. In another study
`[12] TLR2-mediated stimulation of human XLA mononuclear cells
`resulted in impaired production of TNF-α and IL-1β, while production
`of IL-6, IL-8, and IL-10 remained unimpaired [12]. Finally, in a very
`thorough study involving seven XLA patients expressing no Btk protein,
`no effect of Btk deficiency on LPS-induced TNF-α and IL-6 production as
`well as activation of p38 kinase in monocytes was found [13]. Contrary,
`in a murine study Btk-deficient macrophages stimulated with LPS or
`other TLR ligands secreted enhanced levels of IL-6, as a result of
`suppressed IL-10 production [14].
`So far reported, investigations on the role of Btk in TLR signaling
`were carried out in monocytes/macrophages, but not in MCs. Unlike
`macrophages MCs do not express membrane (m)CD14 and differ from
`mCD14-positive macrophages in their LPS recognition properties [15].
`MCs, in the absence of soluble (s)CD14, recognize and react to rough
`(R)-chemotypes of LPS, while the reactivity to smooth (S)-chemotypes
`is practically absent. In this respect, they behave like macrophages
`deficient for CD14 or those expressing a mutant variant of CD14
`(‘heedless’) [16]. In contrast, wild-type macrophages are also able to
`recognize S-chemotypes of LPS in an LPS-binding protein/mCD14-
`dependent fashion [15,16].
`In this work studying the requirement of Btk for LPS- as well as
`lipopeptide (LP) -induced MC activation, we demonstrate that Btk
`moderately inhibits or has no influence on the activation of bone
`marrow-derived MCs (BMMCs) mediated by TLR4 or TLR2. Induction
`of TNF-α and IL-6 by LPS or LP, and activation of p38 kinase, and
`H2O2 production by LPS in Btk-deficient MCs is in comparison to WT
`MCs moderately enhanced or comparable. Interestingly, deficiency of
`PKC-β, which also results in a Xid-like phenotype in mice [17], does
`not influence LPS-induced cytokine production in BMMCs, suggest-
`ing that proteins involved in the organization of the calcium
`signalosome do not, or only marginally participate in TLR4-mediated
`signal transduction in MCs. Finally, Btk plays no obvious role in
`activation of murine bone marrow-derived macrophages (BMMΦs)
`by LPS or LP.
`
`2. Materials and methods
`
`2.1. Reagents
`
`R-form LPS from S. minnesota mutant R595 and S-form LPS from S.
`abortus equi were extracted and purified as described [18–20]. Synthetic
`lipopetides (Pam3CSK4, FSL-1) were obtained from Echaz Microcollec-
`tions (Tübingen, Germany). Polyclonal anti-Mal (T-16) antibody was
`obtained from Santa Cruz (Heidelberg, Germany) and polyclonal anti-
`p85 antibody (#06195) from Upstate/Biomol (Hamburg, Germany).
`Polyclonal anti-P-p38 (T180/Y182) antibody was purchased from Cell
`Signaling Technology (Frankfurt a. M., Germany). DNP-HSA containing
`30–40 mol DNP per mole albumin and monoclonal IgE with specificity
`for DNP (SPE-7) were purchased from SIGMA (Deisenhofen, Germany)
`and DMSO from J.T. Baker (Griesheim, Germany). Btk inhibitor, LFM A13
`[alpha-cyano-beta-hydroxy-beta-methyl-N-(2, 5-dibromophenyl) pro-
`penamide], and cPKC-specific inhibitor, Gö6976, were obtained from
`Calbiochem (Schwalbach, Germany). p38 inhibitor, BIRB0796, was
`purchased from the Division of Signal Transduction Therapy, College
`Of Life Sciences, University of Dundee, Dundee, Scotland, U.K.
`
`2.2. Animals
`
`Breeding of mice and all experiments were done in accordance
`with national guidelines and were approved by a local ethics
`committee.
`
`2.3. Bone marrow-derived mast cells and macrophages
`
`To obtain BMMCs, bone marrow cells (1×106/ml) from 6 to 8 week
`old male mice (129/Sv) were cultured (37 °C, 5% CO2) as a single cell
`suspension in RPMI 1640 medium containing 15% FCS, 1% X63Ag8-
`653-conditioned medium as a source of IL-3 [21], 2 mM L-glutamine,
`1×10− 5 M 2-mercaptoethanol, 50 units/ml penicillin, and 50 mg/ml
`streptomycin. At weekly intervals, the non-adherent cells were
`reseeded at 5 ×105 cells/ml in fresh medium. By 4–6 weeks in culture,
`greater than 99% of the cells were c-kit and FcεR1 positive as assessed
`by phycoerythrin-labeled anti-c-kit antibodies (Pharmingen, Missis-
`sauga, Canada) and FITC-labeled rat anti-mouse IgE antibodies
`(Southern Biotechnology, Birmingham, AL, USA), respectively. Btk+/+
`and −/− MCs were in vitro differentiated using the same protocol but
`starting from BM cells of 6 to 8 week old Btk+/+ and −/− littermates
`(C57BL/6). Wildtype and Xid MCs were in vitro differentiated from BM
`cells of 6 to 8 week old CBA/CaHN-Btkxid/J and CBA/J mice (The Jackson
`Laboratory, Bar Harbor, Maine, USA). PKC-α+/+, PKC-α−/−, PKC-β +/+,
`and PKC-β −/− BMMC were in vitro differentiated from BM cells of 6 to
`8 week old littermates (129/Sv).
`To obtain BMMΦs, BM cells from Btk+/+ and −/− mice were grown
`in the presence of L-cell-conditioned medium in teflon bags as
`described previously [22].
`
`2.4. MC stimulation and Western blotting
`
`IgE-preloaded cells were washed twice in RPMI/0.1% BSA and
`resuspended in RPMI/0.1% BSA. Cells were adapted to 37 °C for 30 min
`and stimulated with the indicated concentrations of DNP-HSA, LPS, or
`LP. After stimulation for different length of time, cells were pelleted
`and solubilized with 0.5% NP-40 and 0.5% deoxycholate in 4 °C
`phosphorylation solubilization buffer [23]. After normalizing for
`protein content, the postnuclear supernatants were subjected
`directly to SDS-PAGE and Western blot analysis as described pre-
`viously [24]. For measurement of extracellular cytokines, cell-free
`supernatants for cytokine measurements were stored in aliquots at
`−80 °C until use.
`
`2.5. Macrophage stimulation
`
`For induction of cytokines, macrophages were resuspended in
`serum-free DMEM (105 cells/0.2 ml/well), placed in 96-well plates
`(Nunc, Roskilde, Denmark) and cultured for 24 h at 37 °C in a
`humidified atmosphere containing 8% CO2. Thereafter macrophages
`were washed and stimulated in triplicates with different amounts of
`LPS or LP in 0.2 ml of DMEM. The culture supernatants for TNF-α and
`IL-6 measurements were collected 4 h and 24 h later, respectively, and
`stored in aliquots at −80 °C until used for cytokine measurements.
`
`2.6. Cytokine measurement
`
`Mouse IL-6 and TNF-α ELISA's (BD Pharmingen, Heidelberg,
`Germany) were performed according to the manufacturer's instruc-
`tions. The levels of cytokines in culture supernatants varied between
`experiments due to genetic background or age of the cells. Qualitative
`differences or similarities between WT and mutant cells, however,
`were consistent throughout the study.
`
`2.7. Flow cytometric analysis of H2O2 production
`
`IgE-sensitized BMMCs were washed twice with PBS, resuspended
`in 500 μl PBS, and stained with H2DCFDA (final concentration: 10 μM)
`for 30 min at 37 °C in the dark. Incubation in the dark was continued
`after the addition of the stimuli (Ag or LPS) for 5 min and flow
`cytometric analysis of cell samples was carried out subsequently
`using a FACScan cell analyzer (Beckton Dickinson, San Jose, USA).
`
`

`

`A
`1800 ,~----,
`D Btk+/+
`■ Btk-/-
`
`1400
`
`l 1000
`
`g
`<D
`...'.J
`
`600
`
`200
`
`B 400
`
`300
`
`l
`g 200
`d
`LL. z
`t-
`
`100
`
`** ,-,
`
`D Btk+/+
`■ Btk-/-
`
`C.N. Zorn et al. / Cellular Signalling 21 (2009) 79–86
`
`81
`
`**
`r-1
`
`Fluorescence intensity data (channel FL1) were collected from 25,000
`cells per measurement.
`
`2.8. RNA extraction and RT-PCR analysis
`
`Total RNA was isolated from 5 × 106 cells by a guanidinium
`isothiocyanate-phenol-chloroform-isoamyl alcohol procedure [25],
`as described previously [26]. Total RNA (1 μg) was reverse-transcribed
`with Moloney Murine Leucaemia Virus reverse transcriptase and
`oligo-p(dT) primers (Expand reverse transcriptase kit; Roche) accord-
`ing to the manufacturer's recommendations. PCR was performed
`using HotStart Taq DNA polymerase (Genaxxon) according to the
`manufacturer's instructions. The primer pairs for amplification
`of murine S0CS1, S0CS3, CISH, and actin were: S0CS1: sense, CCCTGG-
`CGACACTCACTT; antisense, GAGCGCGAAGAAGCAGTT (199-bp product
`size); S0CS3: sense, AGCGTCAAGACCCAGTCG; antisense, ACTTCGGAC-
`GAGGGTTCC (194-bp product size); CISH: sense, TCGGGAATCTGGG-
`TGGTA; antisense, TCCAGCCGGAAGCTAGAA (192-bp product size),
`and actin: sense, TGG AAT CCT GTG GCA TCC ATG AAA; antisense, TAA
`AAC GCA GCT CAG TAA CAG TCC (348-bp product size). An annealing
`temperature of 56 °C was used for actin primer pairs and 61 °C was
`used for S0CS1, S0CS3, and CISH primer pairs. An annealing time of
`25 s was used for S0CS1 as well as actin and of 45 s for S0CS3 and CISH.
`All PCR products were resolved by 2% agarose gel electrophoresis and
`were visualized by ethidium bromide staining.
`
`A
`
`3000
`
`2500
`
`* ,-,
`
`□ Btk+/+
`■ Btk-/-
`
`***
`r-1
`
`0 ..__ ___ ..__
`DNP
`con
`
`0,5
`
`2
`µg/ml LPS
`
`5
`
`C 1500
`
`1000
`
`l g
`
`<D
`...'.J
`
`500
`
`***
`r-1
`
`***
`r-1
`
`0
`
`con
`
`20 ng/ml 0.5µg/ml 2 µg/ml
`DNP
`LPS
`LPS
`
`Fig. 1. Btk is not involved in the positive regulation of LPS-induced cytokine secretion in
`BMMCs. (A) IgE-loaded Btk+/+ and Btk−/− BMMCs were stimulated for 3 h with Ag
`(DNP), increasing concentrations of R-LPS, or left unstimulated (con). Secreted IL-6 was
`measured by ELISA. Each point is the mean of triplicates ±SD. Comparable results were
`obtained with independent BMMC cultures. (B) Supernatants from Btk+/+ and Btk−/−
`BMMCs treated as described in (A) were analyzed by ELISA for the presence of TNF-α.
`Each point is the mean of triplicates± SD. Comparable results were obtained with
`independent BMMC cultures. (C) IgE-loaded WT and Xid BMMCs were treated for 3 h
`with Ag (DNP), increasing concentrations of R-LPS, or left unstimulated (con). IL-6 in the
`supernatants was measured by ELISA. Each point is the mean of triplicates± SD.
`Comparable results were obtained with independent BMMC cultures.
`
`_ 2000
`E
`°' g 1500
`
`<D
`...'.J
`
`1000
`
`500
`
`5
`10 20 0.5
`con DNP 5
`µg/ml FSL-1
`µg/ml Pam3CSK4
`
`***
`;:;:-;
`
`□ Btk+/+
`■ Btk-/-
`
`B 300
`
`=200
`
`t g
`
`d
`LL.
`~ 100
`
`Fig. 2. No attenuation of TLR2-mediated cytokine secretion in Btk-deficient BMMCs.
`(A) IgE-loaded Btk+/+ and Btk−/− BMMCs were stimulated for 3 h with Ag (DNP, 20 ng/
`ml), indicated concentrations of Pam3CSK4 (TLR2/TLR1 ligand) or of FSL-1 (TLR2/TLR6
`ligand), or left unstimulated (con). Secreted IL-6 was measured by ELISA. Each point is
`the mean of triplicates ±SD. Comparable results were obtained with independent
`BMMC cultures. (B) Supernatants from Btk+/+ and Btk−/− BMMCs treated as described
`under (A) were analyzed by ELISA for TNF-α expression. Each point is the mean of
`triplicates± SD. Comparable results were obtained with independent BMMC cultures.
`
`

`

`82
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`C.N. Zorn et al. / Cellular Signalling 21 (2009) 79–86
`
`2.9. Statistical analysis
`
`All values in the figures (concerning bar graphs) are expressed as
`mean of SD of n observations (with n indicated in the respective figure
`
`legends). The relevant data sets were compared by unpaired two
`tailed Student's t test using Graph Pad Prism3 evaluation software
`(Graph Pad, San Diego, CA). P values of ⁎ b0.05, ⁎⁎ b0.005, and ⁎⁎⁎
`b0.0005 were considered statistically significant. Densitometric
`
`A
`Btk-/-
`Btk+/+
`- -- - - - - - - - - - - - - - -- - - - - - -
`DNP
`DNP
`LPS
`LPS
`
`5
`
`15
`
`30
`
`15
`
`30
`
`5
`
`15 30
`
`15
`
`30 min
`
`-----·--- - - _..--1
`WB: o.-P-p38 1--------------·------
`
`WB: o.-p85
`B
`
`□ Btk +/+
`160 ■ Btk -/-
`
`ns
`r7
`
`ns
`r7
`
`120
`
`:::,
`<Ii 80
`
`40
`
`0
`
`C
`
`-
`
`--
`
`WB: o.-actin
`D 3000
`
`= 2000
`~
`3
`(0
`....J
`-
`
`1000
`
`15
`DNP
`Btk +/+
`Pam
`FSL
`DNP
`------
`5 15 30 15 30 15 30
`
`5
`
`30
`
`30
`15
`LPS
`
`min
`
`Btk -/-
`Pam
`FSL
`DNP
`- - - - - -
`15 30 15 30 15 30 min
`
`5
`
`2 93 78 35 52 50 40 38 3 51 27 8 52 48 45 51
`WB: o.-P-p38
`
`--
`
`E
`
`fooNPj
`~
`2000
`
`E
`O>
`3
`~ 1000
`
`7
`
`ODMSO
`■ BI RB0796
`
`0
`
`OM
`
`0,01 0,03 0, 10 0,3
`µM BIRB0796
`
`0 ,....___.L-
`eon DNP FSL Pam LPS
`
`Fig. 3. p38 activation is important for LPS-induced IL-6 secretion, but is not dependent on Btk activity. (A) Btk+/+ and Btk−/− BMMCs were pre-loaded with IgE and stimulated with Ag
`(DNP, 20 ng/ml) or R-LPS (5 μg/ml) for the indicated time points, or were left untreated (−). Subsequently, postnuclear supernatants were analyzed by anti-P-p38 (upper panel) and
`anti-p85 (lower panel; loading control) immunoblotting. (B) Anti-P-p38 Western Blot shown in (A) together with two further experiments performed with independently cultured
`BMMCs were statistically evaluated with respect to changes between Btk+/+ and Btk−/− BMMCs. (C) IgE-preloaded Btk+/+ and Btk−/− BMMCs were stimulated with Ag (DNP, 20 ng/
`ml), FSL-1 (1 μg/ml), or Pam3CSK4 (Pam; 10 μg/ml) for the indicated time points, or were left untreated (−). Subsequently, postnuclear supernatants were analyzed by anti-P-p38
`(upper panel) and anti-actin (lower panel; loading control) immunoblotting. Densitometry was performed, and relative expression levels are indicated under each band. (D) IgE-
`loaded BMMCs were stimulated with Ag (DNP, 20 ng/ml) or R-LPS (5 μg/ml) for 3 h in the presence of the indicated concentrations of p38-specific inhibitor, BIRB0796, or vehicle
`(DMS0, DM). Inhibitor treatment was started 30 min before the addition of stimulus. IL-6 in the supernatants was measured by ELISA. Each point is the mean of triplicates±SD.
`Comparable results were obtained with independent BMMC cultures. (E) IgE-loaded BMMCs were left untreated (con) or stimulated with Ag (DNP, 20 ng/ml), FSL-1 (FSL, 1 μg/ml),
`Pam3CSK4 (Pam, 10 μg/ml), or R-LPS (5 μg/ml) for 3 h in the presence of 0.1 μM BIRB0796 or vehicle (DMS0). Inhibitor treatment was started 30 min before the addition of the stimuli.
`IL-6 in the supernatants was measured by ELISA. Each point is the mean of triplicates± SD. Comparable results were obtained with independent BMMC cultures.
`
`

`

`analysis of Western blots was performed using publicly available
`ImageJ software [27].
`
`A
`
`Btk+/+
`0.5
`
`2
`
`3
`
`0.5
`
`2
`
`3 h
`
`Btk-/-
`
`C.N. Zorn et al. / Cellular Signalling 21 (2009) 79–86
`
`83
`
`3. Results
`
`3.1. Btk deficiency enhances or does not affect LPS- or LP-triggered
`cytokine secretion in BMMCs, respectively
`
`The role of Btk in TLR signaling in MCs was not studied yet. Such
`investigation appeared interesting since activation of MCs via TLR4 or
`TLR2, unlike a similar activation of macrophages, proceeds without
`help of mCD14 and may thus differ from the latter [15]. Here, mast
`cells generated from bone marrow precursors, BMMCs, of wild type
`and Btk-deficient mice were stimulated with LPS in vitro and cytokine
`responses were measured. As shown in Fig. 1A, the levels of IL-6
`secreted by LPS-stimulated Btk−/− and WT BMMCs were either
`comparable or higher in the cultures of Btk−/− cells. Likewise, LPS-
`stimulated Btk−/− BMMCs secreted higher TNF-α levels than WT cells
`(Fig. 1B). However, as reported previously [28], marked reduction of
`secretion of IL-6 and TNF-α was observed in Btk-deficient BMMCs
`compared to WT cells after stimulation via the FcεR1 (Fig. 1A and B).
`Comparable results to those obtained with Btk-deficient BMMCs were
`obtained in studies of IL-6 responses to LPS or Ag of cells derived from
`BtkXid mice (Fig. 1C).
`In this study we also investigated the role of Btk in the IL-6 and TNF-α
`responses of BMMCs to LPs. As shown in Fig. 2A and B, WT and Btk-
`deficient BMMCs stimulated with the synthetic analoga of bacterial
`lipopeptides, FSL-1 (TLR2/TLR6 ligand) and Pam3CSK4 (TLR2/TLR1 ligand),
`secreted moderately enhanced or comparable levels of IL-6 and TNF-α,
`indicating that neither TLR2/TLR6- nor TLR2/TLR1-mediated signaling
`depends on Btk activation in BMMCs.
`
`3.2. TLR-mediated activation of p38 is independent of Btk in BMMCs
`
`In addition to the impaired TNF-α response, impaired activation/
`phosphorylation of p38 kinase was observed earlier in Btk-deficient/-
`compromised monocytes stimulated with LPS [11]. Fitting with our
`data on LPS-induced cytokine secretion in BMMCs, no defect in p38
`phosphorylation was observable in LPS-treated Btk−/− compared to
`WT BMMCs (Fig. 3A and B). Furthermore, also lipopeptide-induced
`p38 phosphorylation was independent of Btk (Fig. 3C). These data
`indicate that in MCs TLR-driven activation of p38 is independent of
`Btk. Ag-triggered p38 activation, however, was severely compromised
`in Btk-deficient BMMCs (Fig. 3A–C). To proof that LPS-induced IL-6
`secretion in BMMCs indeed is dependent on p38 activation, we per-
`formed dose–response analyses with the highly specific p38 inhibitor,
`BIRB0769 [29]. This inhibitor, at the dose of 0.1 μM, blocked both Ag-
`and LPS-triggered IL-6 and TNF-α secretion in BMMCs (Fig. 3D and
`data not shown). Likewise, LP-induced IL-6 and TNF-α responses were
`abrogated by using this inhibitor (Fig. 3E and data not shown). These
`data indicate that TLR-mediated cytokine secretion in BMMCs is, like
`in monocytes, dependent on p38 activation. However, this activation
`is, unlike in monocytes [11], independent of Btk.
`
`3.3. Btk deficiency in BMMCs does neither influence stability of Mal nor
`TLR4-mediated H2O2 production
`
`In macrophages, Btk has been demonstrated to be responsible for
`Mal tyrosine phosphorylation and the subsequent S0CS1/proteasome-
`mediated degradation of this adapter protein [10]. Next, we aimed at
`studying this feedback mechanism in BMMCs. However, we did not
`observe degradation of Mal, neither in WT nor Btk−/− BMMCs, during
`the first 3 h of stimulation (Fig. 4A). This suggests that the respective
`pathway (e.g. S0CS1 expression) is not functional in BMMCs. Indeed,
`performing RT-PCR analysis we observed increased expression of the
`S0CS family member CISH in BMMCs, whereas S0CS1 expression was
`
`WB: a-Mal 1--- -
`
`WB: o.-p85
`
`BMMCs
`.5
`1
`
`2
`
`B
`
`SOCS1
`
`SOCS3
`
`GISH
`
`actin
`
`C
`
`.!!J.
`al
`u
`0
`<ii
`.0
`E
`:::,
`C
`
`BMMCbs
`.5
`1
`I
`
`0
`
`I~ y
`
`11
`
`2
`
`CJJ
`,;;.
`¾
`
`CJJ
`,;;.
`-+-
`
`10° 101
`
`102
`
`103
`
`10° 101
`H20 2
`
`102
`
`103
`
`104
`
`Fig. 4. Btk deficiency does interfere neither with Mal stability nor LPS-induced H2O2
`production. (A) Btk+/+ and Btk−/− BMMCs were stimulated with R-LPS (5 μg/ml) for the
`indicated times or left unstimulated (−). Postnuclear supernatants were subjected to
`Western blot analysis with anti-Mal (upper panel) and anti-p85 (lower panel; loading
`control) antibodies. (B) BMMCs (left panels) and BMMΦs (right panels) were stimulated
`with 10 μg/ml and 1 μg/ml R-LPS, respectively, for the indicated time points. RT-PCR
`analyses were performed using primers for S0CS1, S0CS3, CISH, and actin (loading
`control). Comparable results were obtained with independent BMMC and BMMΦs
`cultures. (C) Btk+/+ (upper panels) and Btk−/− BMMCs (lower panels) were loaded with
`IgE overnight and subsequently incubated for 30 min with the dye H2DCFDA. Cells were
`then either left unstimulated (thin lines) or treated with Ag (DNP, 20 ng/ml; left panels)
`or R-LPS (5 μg/ml; right panels) (thick lines) and H2O2 production analyzed by FACS.
`Comparable results were obtained with independent BMMC cultures.
`
`reduced in response to LPS (Fig. 4B). In contrast, in BMMΦs S0CS1,
`S0CS3, and CISH expression were induced in response to LPS
`stimulation (Fig. 4B).
`Previously, Mangla et al. reported on the positive role of Btk in the
`generation of bursts of reactive oxygen intermediates in macrophages
`[30]. We and others described PI3K-dependent H2O2 production in
`Ag-triggered BMMCs [31,32]. Here we investigated whether LPS is able
`
`

`

`84
`
`C.N. Zorn et al. / Cellular Signalling 21 (2009) 79–86
`
`to stimulate H2O2 production in BMMCs as well and whether this
`process in Ag- or LPS-simulated cells is dependent on Btk. As shown in
`Fig. 4C, both Ag and LPS induced intracellular H2O2 in BMMCs with LPS
`being the weaker stimulus. Interestingly, whereas the Ag-induced
`response was strongly dependent on Btk, LPS-triggered H2O2 produc-
`tion appeared not to be affected by Btk deficiency (Fig. 4C).
`
`3.4. PKC-β deficiency mimics Btk deficiency with respect to LPS- and
`Ag-triggered IL-6 secretion
`
`In addition to Btk deficiency, several deficiencies have been demon-
`strated to result in a Xid-like phenotype, one of them being PKC-β
`deficiency [17]. In agreement with an earlier publication [33], in this study
`we found that Ag-triggered IL-6 production was markedly compromised
`in PKC-β−/− BMMCs, while the production in PKC-α−/−cells was
`unimpaired (Fig. 5A). However, neither PKC-α nor PKC-β single deficiency
`(Fig. 5A), nor PKC-α/PKC-β double deficiency (data not shown), had a
`negative effect on the LPS-triggered IL-6 response of BMMCs. Using an
`inhibitor specific for cPKCs, Gö6976, Ag-triggered IL-6 production
`was almost completely inhibited at a concentration of 0.3 μM, whereas
`
`*** i
`
`D wr
`□ PKC-a-/(cid:173)
`■ PKC-P-/-
`
`A
`7000
`
`5000
`E
`O> s
`~3000
`
`a
`
`LPS
`
`1000 -
`
`■
`O con DNP
`
`B 250
`
`200
`
`E 150
`O>
`8
`<O
`...'.J 100
`
`50
`
`O DMSO
`
`0.1
`0.3
`- - - - -
`µM Go6976
`
`Fig. 5. PKC-β-deficiency results in compromised IL-6 production after Ag treatment, but
`normal response to LPS. (A) WT, PKC-α−/−, and PKC-β−/− BMMCs were left untreated
`(con) or stimulated for 3 h with Ag (DNP, 20 ng/ml), or R-LPS (2 μg/ml). Subsequently, IL-
`6 in the supernatants was measured by ELISA. Each point is the mean of triplicates± SD.
`Comparable results were obtained with independent BMMC cultures. (B) IgE-loaded
`BMMCs were stimulated with Ag (DNP, 20 ng/ml) or R-LPS (2 μg/ml) for 3 h in the
`presence of vehicle (DMS0) or the indicated concentrations of the cPKC inhibitor,
`Gö6976. Inhibitor treatment was started 30 min before the addition of the stimuli. IL-6
`in the supernatants was measured by ELISA. Each point is the mean of triplicates± SD.
`
`**
`
`D DMSO
`CLFM A13 10 µM
`■ LFM A13 100 µM
`***
`r.;-,
`
`4000
`
`= 3000
`~
`.3
`~ 2000
`
`1000
`
`OJ...J.. ...... -..1--....1....1..---..;;;;;,-....._ ..................... . - - -
`con DNP LPS
`con DNP LPS
`Btk +/+
`Btk -/ -
`
`Fig. 6. LFM A13 exerts off-target effects in Btk-deficient BMMCs. IgE-loaded Btk+/+ and
`Btk−/− BMMCs were left untreated (con) or stimulated with Ag (DNP, 20 ng/ml) or R-LPS
`(2 μg/ml) for 3 h in the presence of vehicle (DMS0), 10 μM LFM A13, or 100 μM LFM A13.
`Inhibitor treatment was started 30 min before the addition of stimulus. IL-6 in the
`supernatants was measured by ELISA. Each point is the mean of triplicates± SD.
`
`LPS-induced IL-6 secretion was only marginally affected (Fig. 5B). This
`indicates that in BMMCs neither PKC-β- nor Btk deficiency exerts a
`negative impact on LPS-induced cytokine production. Moreover, this
`suggests that different deficiencies resulting in a Xid-like phenotype do
`not abrogate TLR-mediated BMMC activation.
`
`3.5. LFM A13 blocks LPS-induced IL-6 production even in Btk-deficient
`BMMCs
`
`In different studies, the Btk-specific inhibitor, LFM A13, has been
`used to show Btk dependence of TLR-mediated signal transduction
`[8 –10]. However, the potential off-target effect of this drug was not
`investigated in Btk-deficient or -mutant cells. Therefore, we measured
`IL-6 production in WT as well as Btk-deficient BMMCs treated with
`different concentrations of LFM A13 (10 and 100 μM) and subse-
`quently stimulated with Ag or LPS. As shown in Fig. 6, LFM A13 at
`100 μM, a concentration range used by others [8–10,34], completely
`inhibited both Ag- and LPS-triggered IL-6 secretion in both WT and
`Btk-deficient BMMCs. Whereas 10 μM LFM A13 did not show any effect
`on the Ag-induced IL-6 production, it slightly attenuated LPS-induced
`production in WT and even stronger in Btk−/− BMMCs (Fig. 6). This
`clearly indicates that the inhibitory effect of LFM A13 on IL-6 produc-
`tion of MCs is not a result of Btk blockade. Therefore, LFM A13 cannot
`be considered a Btk-specific inhibitor, and that data on the role of
`Btk obtained solely by the usage of this drug might very likely be
`erroneous.
`
`3.6. Btk deficiency does not impair LPS or LP-triggered cytokine secretion
`in bone marrow-derived macrophages
`
`Conflicting results were obtained when the role of Btk for TLR
`signaling was studied in human monocytes or mouse macrophages
`[11–13]. Since we found Btk to be dispensable for TLR-mediated acti-
`vation of MCs derived from mouse bone marrow, we finally analyzed
`the role of Btk in the TLR-triggered induction of cytokines in macro-
`phages grown in vitro from murine bone marrow precursors. For
`this purpose, Btk+/+ and Btk−/− BMMΦs were stimulated with R- and
`S-chemotypes of LPS, as well as with the LPs, FSL-1 and Pam3CSK4, and
`production of IL-6 (Fig. 7A) and TNF-α (Fig. 7B) was measured. In
`agreement with the data by Perez de Diego [13], but contrary to reports
`demonstrating abrogated TLR responses of Btk-deficient macrophages
`[11,12], the LPS- and LP-induced cytokine production was indistin-
`guishable between WT and Btk-deficient BMMΦs. These data in
`
`

`

`C.N. Zorn et al. / Cellular Signalling 21 (2009) 79–86
`
`85
`
`A
`
`□ Btk+/+
`7 ■ Btk-/-
`
`* r-=
`
`**
`r-=
`
`**
`r-=
`
`0
`
`con
`
`B
`
`3
`
`0 Btk+/+
`■ Btk-/-
`
`I
`
`£2
`t:l
`LL
`z
`I-
`
`1
`
`Pam3CSK4
`
`* r-=
`
`* r-=
`
`0
`
`con
`
`1 10 100ng/ml
`Pam3CSK4
`
`Fig. 7. Btk-deficient BMMΦs are not compromised in cytokine production in response to
`different TLR ligands. (A) Btk+/+ and −/− BMMΦs were left untreated (con) or stimulated
`for 24 h with the indicated concentrations of R-LPS, S-LPS, FSL-1, or Pam3CSK4. IL-6 in
`the supernatants was measured by ELISA. Each point is the mean of triplicates± SD.
`(B) Btk+/+ and −/− BMMΦs were left untreated (con) or stimulated for 4 h as in A. TNF-α
`in the supernatants was measured by ELISA. Each point is the mean of triplicates± SD.
`Each point is the mean of triplicates± SD. Comparable results were obtained with
`different BMMΦ cultures.
`
`combination with the published reports suggest that the source and
`differentiation conditions of macrophages are decisive for their
`dependence on Btk for TLR signaling.
`
`4. Discussion
`
`Several research groups reported on the role of Btk in TLR signaling
`in monocytes/macrophages of murine as well as human origin
`[8,10–14].
`In this study,
`loss of Btk function either moderately
`enhanced (LPS triggering) or did not influence (LP triggering) TLR4 and
`TLR2 mediated responses of mouse BMMCs. Thus, in MCs Btk exerts a
`weak inhibitory function on TLR4 signaling and has no obvious effect
`on TLR2 signaling. Earlier, Btk was ascribed a negative regulatory role
`in activation of mouse dendritic cells, mainly through the autocrine
`secretion of IL-10 [35]. Accordingly, Btk−/− DCs produce less IL-10 in
`response to LPS [35]. This mechanism, however, seems to be absent in
`BMMCs, since in preliminary experiments we were unable to detect
`LPS-induced IL-10 production in BMMCs (data not shown). Moreover,
`in another study, production of IL-10 and IL-12p70 was comparable
`between DCs from healthy donors and XLA patients [34].
`We also show in this study (Fig. 7) that macrophages differentiated
`from WT or Btk−/− murine bone marrow cells do not exert differences
`in TLR-mediated production of pro-inflammatory cytokines although
`such differences were reported by others [11,12,14]. One important
`
`question in light of all these apparent discrepancies is how can such
`significant differences between