Bmx tyrosine kinase regulates TLR4-induced IL-6 production in human macrophages independently of p38 MAPK and NF B activity
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CHEMOKINES, CYTOKINES, AND INTERLEUKINS
Bmx tyrosine kinase regulates TLR4-induced IL-6 production in human
macrophages independently of p38 MAPK and NFB activity
Christine D. Palmer,1 Brenda E. Mutch,1 Sarita Workman,2 John P. McDaid,1 Nicole J. Horwood,1 and Brian M. J. Foxwell1
1KennedyInstitute of Rheumatology Division, Faculty of Medicine, Imperial College London, London; and 2Royal Free Medical School, Department of
Immunology, University College London, London, United Kingdom
Chronic inflammation, as seen in condi- RNA interference results in decreased gets the IL-6 3ⴕ untranslated region to
tions such as rheumatoid arthritis and tumor necrosis factor-␣, but not IL-6 pro- increase mRNA stabilization via a novel,
Crohn disease, is in part driven by discor- duction. Further investigations into the thus far undefined, p38 mitogen activated
dant production of inflammatory cyto- signaling mechanisms regulating IL-6 pro- protein kinase-independent pathway.
kines, such as tumor necrosis factor-␣ duction led to the discovery that the Tec These data have important implications
and interleukin-6 (IL-6). Tyrosine kinase kinase bone marrow tyrosine kinase gene for the design of therapeutics targeted
activity is essential to lipopolysaccharide- in chromosome X (Bmx) regulates Toll- against specific cytokines and their regu-
induced cytokine production in mono- like receptor-induced IL-6 production. Our lators in inflammatory disease. (Blood.
cytes, and previous studies by us and data further showed that Bmx-dependent 2008;111:1781-1788)
others have implicated a role for the Tec super-induction of IL-6 does not involve
kinase Bruton’s tyrosine kinase (Btk) in nuclear factor–B activity. More detailed
inflammatory cytokine production. Here investigations of pathways downstream
we show that knockdown of Btk using of Bmx signaling revealed that Bmx tar- © 2008 by The American Society of Hematology
Introduction
The discovery of Toll-like receptors (TLR) was a major advance in conclusions,10,11 whereas others ruled out a role for Pyk2 and Syk
our understanding of host responses to infection.1 Detection of in LPS-induced cytokine production.12,13 Recently, studies from
pathogen-associated molecular patterns, such as lipopolysaccha- our laboratory and others have shown that the Tec kinase Bruton’s
ride (LPS) by TLRs, leads to the activation of immune cells, tyrosine kinase (Btk) is involved in TLR signaling.14-17 The Tec
particularly macrophages and other cells of the myeloid lineage.2 family tyrosine kinases are structurally similar to the Src kinases,
One of the most potent effects of the TLR activation is the and 3 of its members are expressed in human monocytes and
induction of inflammatory cytokines, such as tumor necrosis macrophages: tyrosine kinase expressed in hepatocellular carci-
factor␣ (TNF), interleukin-1 (IL-1), and IL-6.3 The realization that noma (Tec), bone marrow tyrosine kinase gene in chromosome X
these cytokines play a key role in chronic inflammatory diseases, (Bmx), and Btk. Btk is associated with the human immunodefi-
such as rheumatoid arthritis (RA) and Crohn disease, has focused ciency X-linked agammaglobulinemia (XLA),18,19 and using XLA
interest onto the molecular mechanisms that regulate the produc- monocytes, we showed that Btk deficiency leads to reduced
tion of inflammatory cytokines. This has gained particular impetus TLR-induced TNF and IL-1 production, whereas the production of
from the discovery that TLRs may also be involved in regulating/ IL-6 was unaffected.14,17 This was unexpected because IL-6
perpetuating chronic inflammatory conditions through the recogni- production is also blocked by tyrosine kinase inhibitors.8,9 Addi-
tion of endogenous ligands produced as a result of tissue injury.4 tional studies using overexpression of Btk in wild-type human
TLR engagement leads to the activation of numerous signaling macrophages showed that Btk regulated TNF mRNA stability
pathways regulating cytokine production, including the nuclear through a pathway involving p38 MAPK, rather than transcription.14 In
factor (NF)–B family of transcription factors and the mitogen contrast, investigations in transformed cell lines using a dominant
activated protein kinase (MAPK) p38.5,6 negative construct and Btk-deficient murine macrophages suggested
Tyrosine phosphorylation is detectable almost immediately that Btk signaled upstream of NF-B, and presumably TNF transcrip-
after LPS stimulation in murine macrophages,7 and studies using tion rather than p38 MAPK signaling.15,16 Studies by other groups
broad range tyrosine kinase inhibitors in human blood monocytes have since shown no change in TNF expression in XLA monocytes,20
demonstrated that tyrosine phosphorylation is required for TNF, defects in XLA dendritic cell signaling via TLR8,21 and impairment
IL-6, and IL-1 production in response to LPS.8,9 Despite this, the of IL-10 expression in murine cells.22 Thus, the involvement of
involvement of tyrosine kinases in TLR signaling remains unclear. Btk in TLR-induced cytokine production in human and murine cells
Several protein tyrosine kinases are activated by LPS, including remains controversial.
Src family kinases, the focal adhesion kinase Pyk2, and Syk kinase. To address these discordant findings, we generated Btk-
Previous investigations into the Src kinases led to conflicting depleted human macrophages using RNA interference (RNAi)
Submitted July 23, 2007; accepted October 21, 2007. Prepublished online as Blood The publication costs of this article were defrayed in part by page charge
First Edition paper, November 19, 2007; DOI 10.1182/blood-2007-07-102343. payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement. © 2008 by The American Society of Hematology
BLOOD, 15 FEBRUARY 2008 䡠 VOLUME 111, NUMBER 4 1781From www.bloodjournal.org by guest on September 8, 2015. For personal use only.
1782 PALMER et al BLOOD, 15 FEBRUARY 2008 䡠 VOLUME 111, NUMBER 4
knockdown, thereby reducing the chance of compensating changes previously described.14 The NF-B luciferase adenovirus (AdNFB-luc)
in other genes, effects of transformation, or variations in the nature contains 4 tandem copies of the enhancer element located upstream of the
of Btk mutations. These studies confirmed our previous findings firefly luciferase gene.6 This adenovirus was provided by P. B. McCray Jr
that Btk contributes to the regulation of TNF production after TLR (University of Iowa, Iowa City, IA) and is a modification of the pNFB
reporter vector (BD Biosciences UK, Oxford, United Kingdom). M-CSF-
stimulation, whereas IL-6 production remained unchanged.17 These
derived macrophages were plated in a 96-well plate at 105 cells/well and
data further substantiated the differential regulation of TNF and allowed to express adenoviral transgenes for at least 24 hours before
IL-6 after TLR stimulation, which seems to be in stark contrast to stimulation with LPS as previously described.14 For luciferase assays, cells
early studies using broad-spectrum tyrosine kinase inhibitors in were subjected to 2 subsequent rounds of infections: first with the WT
human monocytes showing clearly that LPS-induced IL-6 produc- adenoviral constructs, followed by a 2-hour recovery period in complete
tion depends on tyrosine kinase activity.9 This led us to investigate medium, and second with luciferase adenoviral constructs 24 hours
whether the closely related Tec kinase Bmx might be involved in before stimulation.
regulating IL-6 production after stimulation with LPS. Here we
show that, unlike its family members Btk and Tec, Bmx is RNAi
expressed in undifferentiated XLA and control peripheral blood For targeted protein knockdown using RNAi, 5 ⫻ 106 freshly elutriated
mononuclear cells (PBMCs) and monocytes. These cells produce human monocytes were transfected with targeting siRNA or control
normal levels of IL-6, and we therefore went on to investigate a oligunucleotides (siControl D-001206-13 and human Btk SMARTpool
possible role for Bmx in TLR-induced cytokine production. This M-003107-01, Dharmacon, IL) at concentration ranges from 100 to
study shows, for the first time, that Bmx is activated in human 300 nM using the Human Monocyte Nucleofector Kit (Amaxa Biosystems,
macrophages stimulated with LPS and that Bmx shows partial Cologne, Germany) according to the manufacturer’s instructions. After
overlap with Btk in the regulation of TNF, and a parallel but distinct transfection, monocytes were differentiated in the presence of M-CSF as
mechanism regulating IL-6 independently of both p38 MAPK and described in “Isolation of monocytes by elutriation and culture of macro-
phage colony-stimulating factor–derived macrophages.” Protein knock-
NF-B activity in primary human macrophages.
down was subsequently assessed by Western blotting.
Enzyme linked immunosorbent sandwich assay
Methods
The concentrations of TNF, IL-6, IL-8, and IL-10 were determined by
Reagents ELISA (BD PharMingen, San Diego, CA) according to the manufacturer’s
instructions. Absorbance was read and analyzed at 450 nm on a spectropho-
LPS, Pam3Cys-SerLys4 (Pam3C-SK4), Malp-2, and Flagellin were ob- tometric enzyme-linked immunosorbent sandwich assay (ELISA) plate
tained from Alexis Biochemicals (Nottingham, United Kingdom). SB203580 reader (Labsystems Multiskan Biochromic; Labsytems, Basingstoke, United
and proteasome inhibitor I (PSI) were obtained from Calbiochem (Notting- Kingdom) using the Ascent software program.
ham, United Kingdom). All reagents (other than LPS) were tested for the
presence of endotoxin using the Limulus amoebocyte assay (BioWhittaker Western blotting
UK, Wokingham, United Kingdom). Optimal dose ranges for the use of
TLR ligands in primary macrophages have been assessed previously.14,17 M-CSF-derived macrophages were plated in a 12-well plate at 106 and
either left untreated or infected with adenovirus constructs as before and
Isolation of monocytes by elutriation and culture of allowed to express adenoviral transgenes for 24 hours before stimulation.
macrophage colony-stimulating factor–derived macrophages Whole cell protein extracts were prepared as previously described.14
Samples were resolved by 8% SDS-PAGE. Primary antibodies used to
PBMCs were prepared from single donor blood buffy coat fractions using visualize the samples were mouse anti-Btk (BD Biosciences) rabbit
Ficoll-Hypaque centrifugation, and monocytes were then isolated by anti-Bmx (a gift from Michael G. Tomlinson), rabbit anti-Tec (Upstate
centrifugal elutriation, as previously described.23 Monocyte fractions of Biotechnology, Charlottesville, VA), mouse anti-HA (Covance Research
80% purity or higher were routinely collected and cultured in RPMI Products, Princeton, NJ), and mouse anti–␣-tubulin (Sigma Chemical,
containing 10% heat inactivated fetal calf serum at 37°C in a humidified Poole, United Kingdom). Secondary antibodies used were sheep antimouse
atmosphere containing 5% CO2. For adenoviral infection, monocytes were and donkey antirabbit (GE Healthcare, Chalfont St Giles,
treated with 100 ng/mL macrophage colony-stimulating factor (M-CSF; United Kingdom).
Genetics Institute, Cambridge, MA) for 96 hours before infection.
Immunoprecipitation and kinase assay
Isolation and culture of PBMCs from XLA patients and
control donors M-CSF macrophages were plated into 10-cm2 cell culture dishes at a
density of 5 ⫻ 106 cells/dish and infected with HA-tagged Bmx as
Human blood samples were collected into lithium heparin vacutainers and described in “Western blotting.” After 24 hours, cells were washed and
PBMCs isolated as previously described.14 PBMCs were cultured at a rested in serum-free medium for 2 hours. After stimulation with LPS, cells
concentration of 106 cells/mL in RPMI containing 100 units/mL penicillin/ were subjected to immunoprecipitation and auto-kinase assay as previously
streptomycin and 10% heat-inactivated fetal calf serum at 37°C in a described.14,17 Radiolabeled species were visualized by autoradiography
humidified atmosphere containing 5% CO2. Ethical permission for the using Hyperfilm (GE Healthcare).
study was obtained from the Royal Free Hospital and Medical School
research ethics committee, and written informed consent was obtained from Real-time PCR
all patients in accordance with the Declaration of Helsinki.
M-CSF-derived macrophages were plated in a 24-well plate at 5 ⫻ 105 and
Generation of adenoviral vectors and cell infection infected as described in “Generation of adenoviral vectors and cell
infection.” Cells were treated with LPS for 4 hours before the addition of
Recombinant, replication-deficient adenoviral constructs encoding wild- actinomycin D (2 g/mL; Sigma Chemical) and harvested at 0, 15, 30, 60,
type human Bmx and HA-tagged Bmx (AdBmx and AdBmxHA) were 90, and 120 minutes. Total RNA was extracted using RNeasy Kit
prepared using the AdEasy system as previously described.14,24 pAdTrack- (QIAGEN, Dorking, United Kingdom) according to the manufacturer’s
IL-6 5⬘ promoter-Luc-3⬘untranslated region (UTR; AdIL-6 5⬘/3⬘) and instructions. All semi-quantitative RT-PCR was performed as previously
pAdTrack-IL-6 5⬘ promoter-Luc (AdIL-6 5⬘only) were generated as described.17 All quantifications were normalized to an endogenous control,From www.bloodjournal.org by guest on September 8, 2015. For personal use only.
BLOOD, 15 FEBRUARY 2008 䡠 VOLUME 111, NUMBER 4 Bmx REGULATES IL-6 PRODUCTION IN MACROPHAGES 1783
Figure 1. Targeted RNAi knockdown of Btk protein results in decreased TNF, not IL-6, production in human macrophages after LPS stimulation. Peripheral blood
monocytes were transfected with increasing doses of targeting and control siRNA oligonucleotides and differentiated in the presence of 100 ng/mL of M-CSF in culture for
4 days. (A) The expression of Btk was assessed by Western blotting. Densitometry units (mean ⫾ SEM) for 4 separate donors are shown normalized to untreated controls.
(B) For analysis of cytokine expression, siRNA-transfected M-CSF macrophages were treated with LPS (10 ng/mL) for 18 hours and supernatants assessed for TNF and IL-6
levels by ELISA. Values are shown as mean plus or minus SEM for 4 separate donors normalized to LPS only controls (**P ⬍ .01). (C) PBMCs were prepared from XLA and
normal male donors (age range, 17-46 years) as described in “Isolation and culture of PBMCs from XLA patients and control donors.” Cells were left undifferentiated (XLA f,
normal F) or cultured in the presence of M-CSF (100 ng/mL) for 4 days (XLA 䡺, normal E) and then stimulated with LPS (10 ng/mL) or Pam3C-SK4 (100 ng/mL). Cytokine
production was assessed by ELISA 18 hours after stimulation. Each data point shown represents a single donor (n.s., not significant). (D) Bmx and Tec protein expression in
matched undifferentiated and M-CSF-treated XLA PBMCs, normal PBMCs, and blood monocytes was assessed by Western blotting. Blots are representative of 4 separate
donors for each matched cell population. Statistical significance was assessed using one-way ANOVA and Bonferroni multiple comparison test.
the housekeeping gene GAPDH, to account for variability in the initial
concentration of RNA and the conversion efficiency of the reverse
transcription reaction. The analysis of the relative quantitation required
calculations based on the threshold cycle (Ct); the cycle number at which
the amplification plot crosses a fixed threshold above baseline is defined as
Ct. Relative quantitation was performed using the comparative ⌬⌬Ct
method according to the manufacturer’s instructions.
Luciferase reporter gene assay
After LPS stimulation, cells were washed once in phosphate-buffered
saline and lysed with 100 L CAT lysis buffer. Cell lysates (50 L) were
transferred into a luminometer cuvette strip and 120 L luciferase assay
buffer and 30 L luciferin added as described previously.14 Luciferase
activity was measured in relative luciferase units (RLU) using the
PerkinElmer Wallac MicroBeta Tri Lux luminometer (GMI) machine
and software.
Electrophoretic mobility shift assay
Electrophoretic mobility shift assay (EMSA) was performed on nuclear
extracts from M-CSF macrophages, which were infected with AdBmx and
Figure 2. LPS stimulation induces Bmx kinase activity in primary human macro-
control virus as described in “Generation of adenoviral vectors and all
phages. PBMCs were differentiated into macrophages in the presence of 100 ng/mL of
infection.” Nuclear extracts were prepared as described previously.6 The M-CSF for 4 days. Cells were infected for 2 hours with adenoviruses overexpressing
extract was optimized for uniform quantitative loading of protein (5 g) HA-tagged wild-type Bmx or control adenovirus Ad0 at a multiplicity of infection of 100:1 in
using the BCA kit for protein estimation (Pierce Biotechnology; Rockford, serum-free medium. (A) Expression of HA-Bmx at different multiplicities of infection was
IL) and DNA binding activity assessed by EMSA as described previously.6 assessed by Western blotting. (B) For auto-kinase assay, cells were cultured in complete
Gels were dried on chromatography paper (Schleicher & Schuell, London, medium and treated with 10 ng/mL of LPS for 0, 5, 10 and 20 minutes. Cells were lysed and
HA-Bmx immunoprecipitated from lysates as described in “Immunoprecipitation and
United Kingdom) and exposed to high performance chemiluminescence
kinase assay” and subjected to in vitro kinase assay. (C) Densitometry units (mean ⫾ SEM)
film (Hyperfilm ECL, GE Healthcare) at ⫺70°C, and phospho imaged pooled for 3 separate donors are shown normalized to untreated controls. Statistical
using an AGFA Curix 60 Film Processor (Agfa/Gevaert, Brentford, significance was assessed using one-way ANOVA and Bonferroni multiple comparisons
United Kingdom). test (**P ⬍ .01).From www.bloodjournal.org by guest on September 8, 2015. For personal use only.
1784 PALMER et al BLOOD, 15 FEBRUARY 2008 䡠 VOLUME 111, NUMBER 4
Figure 3. Bmx regulates TLR-induced TNF and IL-6 production.
Macrophages were infected with AdBmx or Ad0 as before. Cells
were cultured in complete medium and stimulated with LPS
(10 ng/mL), Pam3C-SK4 (100 ng/mL), Malp2 (100 ng/mL), or
Flagellin (100 ng/mL) for 18 hours. Cytokine expression in superna-
tants was assessed by ELISA. (A) LPS-induced TNF, IL-6, IL-8, and
IL-10 production was assessed in uninfected, Ad0-infected, and
AdBmx-infected macrophages. Values (mean ⫾ SEM) for 4 sepa-
rate donors normalized to LPS-treated controls are shown.
(B) Cytokine production after Pam3C-SK4, Malp2, and Flagellin
stimulation was assessed by ELISA. Values (mean ⫾ SEM) for 4
separate donors normalized to Pam3C-SK4- and Malp-2-treated
controls are shown. Statistical significance was assessed using
Student t test (*P ⬍ .05; **P ⬍ .01; ***P ⬍ .001).
SK4- or LPS-induced IL-6 production in XLA and control PBMCs
Results showed no changes after M-CSF differentiation (Figure 1C).
Parallel studies using normal monocytes, PBMCs, and XLA
Targeted knockdown of Btk protein using RNAi results in PBMCs showed a constant expression of Bmx in all 3 cell
reduced TNF, but not IL-6, production populations (Figure 1D). These findings indicate that Tec is not
involved in TLR-induced IL-6 production in undifferentiated cells.
Given that our previous data from XLA cells had shown a defect in
LPS-induced TNF, but not IL-6 production, we investigated
whether RNAi knockdown of Btk would produce a similar result. LPS activates Bmx kinase activity in human
Transfection of human macrophages with siRNA targeting Btk M-CSF macrophages
resulted in dose-dependent knockdown of Btk protein compared
with controls (Figure 1A). Suppression of Btk expression resulted Given the correlation between the constant expression of Bmx with
in a significant decrease in LPS-induced TNF expression, with IL-6 the unaffected production of IL-6, we questioned whether there
production remaining unchanged (Figure 1B). could be a causal link between the two. We used adenoviruses
encoding wild-type Bmx (AdBmx) and HA-tagged Bmx (AdBmx
Bmx protein expression and IL-6 production are independent of HA) to infect human macrophages at increasing multiplicity of
M-CSF differentiation infection. HA-tagged Bmx protein levels increased dose-
dependently from 50:1 to 150:1 multiplicity of infection in
Previous studies showed that Btk and Tec expression levels were macrophages, with no increase in Bmx expression observable in
low in undifferentiated human PBMCs and monocytes but up- Ad0-infected controls (Figure 2A). Similar data were obtained for
regulated with M-CSF differentiation.14 We therefore investigated untagged Bmx (data not shown). In the absence of precipitating
whether TLR-induced IL-6 production differed between undifferen- antibodies to Bmx, we used HA-tagged Bmx to ascertain changes
tiated and M-CSF-treated XLA PBMCs. Studies examining Pam3C- in kinase activity in response to LPS stimulation in humanFrom www.bloodjournal.org by guest on September 8, 2015. For personal use only.
BLOOD, 15 FEBRUARY 2008 䡠 VOLUME 111, NUMBER 4 Bmx REGULATES IL-6 PRODUCTION IN MACROPHAGES 1785
Figure 4. Bmx does not signal upstream of NF-B. Macrophages were infected for 2 hours with adenoviruses overexpressing wild-type Bmx or control adenovirus Ad0 in
serum-free medium. For luciferase assays, cells were subjected to a second round of infections with NF-B consensus luciferase adenovirus (AdNFB-luc) before stimulation.
Cells were stimulated with 10 ng/mL LPS for 18 hours for ELISA, 4 hours for luciferase reporter gene assays, and 0.5 and 1 hour for EMSA. (A) Uninfected, Ad0-infected, and
AdBmx-infected macrophages were left untreated or preincubated with PSI (1 M) and TNF and IL-6 production assessed by ELISA. Values (mean ⫾ SEM) are presented as
percent compared with LPS-treated uninfected controls (100%; *P ⬍ .05; **P ⬍ .01). (B) Relative luciferase units (RLU) for uninfected, Ad0-infected, and AdBmx-infected cells
in the absence (left hand panel) or presence (right hand panel) of AdNFB-luc after 4 hours of LPS stimulation are shown as mean (⫾ SEM) for
4 separate donors normalized to LPS only controls. n.s, not significant. (C) Nuclear extracts were collected as described in “Electrophoretic mobility shift assays” and assayed
for NF-B DNA binding by EMSA. Statistical significance was assessed by Student t test.
macrophages. As demonstrated in Figure 2B, Bmx auto-kinase struct (Figure 4B). Additional studies by EMSA showed that
activity was increased within 5 minutes in the presence of LPS. overexpression of Bmx did not increase LPS-induced binding of
Densitometric analysis pooled for 3 separate donors showed a rapid the NF-B consensus oligonucleotide (Figure 4C).
increase in Bmx auto-kinase activity within 5 minutes of LPS
stimulation, which returned to just above base level at 10 minutes
Bmx induces TNF and IL-6 mRNA stabilization via 2
after stimulation (Figure 2C).
separate pathways
Overexpression of Bmx enhances TLR-induced TNF and IL-6, LPS-induced TNF expression is regulated posttranscriptionally via
but not IL-8 or IL-10 production mRNA stabilization, a process that involves the MAPK p38 and
targeting of the 3⬘UTR of the TNF gene.26-29 Therefore, we
Infection of macrophages with wild-type AdBmx resulted in a 2- to
investigated possible involvement of p38 MAPK downstream of
3-fold increase in levels of TNF in response to LPS stimulation
Bmx signaling. The p38 MAPK inhibitor SB203580 ablated the
compared with uninfected and control infected cells (Figure 3A).
enhancing effect of AdBmx on LPS-induced TNF production
Wild-type Bmx overexpression also increased IL-6 production by
(Figure 5A). In contrast, SB203580 did not affect IL-6 production
2- to 3-fold but did not affect the production of IL-8 and IL-10 in
in any circumstance (Figure 5A). Studies in RAW 264.7 murine
response to LPS stimulation (Figure 3A). Similarly, Bmx overex-
macrophages have suggested a role for the MAPK c-Jun N-
pression also resulted in increased TNF and IL-6, but unchanged
terminal kinase (JNK) in translational regulation of LPS-induced
IL-8 and IL-10, production in macrophages stimulated with TLR
TNF production.30 We thus investigated whether JNK and the other
ligands Pam3C-SK4 (TLR1/2), Malp-2 (TLR2/6), and Flagellin
MAP kinase, extracellular signal-related protein kinase (ERK),
(TLR5; Figure 3B).
may have a role in LPS-induced IL-6 production. However, neither
Bmx-dependent IL-6 super-induction does not involve the ERK inhibitor PD9805931 nor the JNK inhibitor SP60012532
NF-B activity had any effect on LPS induced IL-6 production (data not shown),
suggesting that, in contrast to TNF, ERK, and JNK MAPK are not
LPS-induced production of IL-6 in macrophages depends on involved in translational regulation of IL-6. We subsequently
activation of the NF-B,25 and treatment with PSI decreased investigated the effect of Bmx overexpression on TNF and IL-6
LPS-induced TNF and IL-6 production by 40% and 80%, respec- mRNA stability using actinomycin D chase studies. Bmx overex-
tively, but had no effect on the enhancement induced by AdBmx pression increased TNF mRNA stability compared with controls
(Figure 4A). Our hypothesis that Bmx regulates IL-6 production (Figure 5B). However, the Bmx effect was not restricted to TNF
independently of NF-B activation was further supported by because there was also stabilization of IL-6 mRNA. Again, the role
studies showing that overexpression of Bmx had no effect on the of p38 MAPK in this process was investigated using SB203580,
LPS response of an NF-B–driven luciferase reporter gene con- which abrogated Bmx-induced TNF mRNA stabilization, but didFrom www.bloodjournal.org by guest on September 8, 2015. For personal use only.
1786 PALMER et al BLOOD, 15 FEBRUARY 2008 䡠 VOLUME 111, NUMBER 4
A Figure 5. Bmx increases TNF and IL-6 mRNA stabilization via
2 distinct downstream pathways. (A) Macrophages were left
200 * 300 no virus uninfected or infected with Ad0 and AdBmx as before and left
Percent Induction TNF
*
Percent Induction IL-6
Ad0 untreated or preincubated with SB203580 (1 M). TNF and IL-6
150 *** AdBmx production was assessed by ELISA. Values (mean ⫾ SEM) are
200
presented as percent compared with LPS-treated uninfected
100 controls (100%). Statistical significance was assessed using
n.s. 100 Student t test. n.s., not significant (*P ⬍ .05; ***P ⬍ .001).
50 (B) Macrophages were left uninfected or infected with Ad0 and
AdBmx as before. Cells were stimulated with LPS (10 ng/mL) for
0 0 4 hours before addition of actinomycin D (2 g/mL). TNF and IL-6
Control SB203580 Control SB203580
mRNA levels at 0, 15, 30, 60, 90, and 120 minutes after addition of
actinomycin D were assessed by real-time PCR. Values
B (mean ⫾ SEM) for 4 separate donors are shown normalized
Uninfected
1000 1000 to time 0 of actinomycin D addition (100%) (**P ⬍ .01).
Percent IL-6 mRNA
Percent TNF mRNA
Ad0
AdBmx (C) Macrophages were left uninfected or infected with Ad0 and
AdBmx as before and left untreated or preincubated with SB203580
100 ** 100
** (1 M). TNF and IL-6 mRNA levels at 1 and 2 hours after addition
of actinomycin D in the presence or absence of SB203580 (1 M)
10 10
were assessed by real-time PCR. Values (mean ⫾ SEM) for
4 separate donors are shown normalized to time 0 of actinomycin
1 1 D addition (100%). Statistical significance was assessed by
0 15 30 45 60 75 90 105120 0 15 30 45 60 75 90 105120 one-way ANOVA and Bonferroni multiple comparison test
Minutes Minutes (*P ⬍ .05; **P ⬍ .01).
C
TNF IL-6 no virus
TNF mRNA Percent Induction
Ad0
IL-6 mRNA Percent Induction
AdBmx
1000 1000 * ** * *
**
**
100 100
10 10
+ + + + + LPS (4hr) + + + + + LPS (4hr)
0 1 1 2 2 A ctD (hr) 0 1 1 2 2 A ctD (hr)
- - + - + SB (1 M ) - - + - + SB (1 M )
not alter Bmx-induced IL-6 mRNA stabilization after treatment using both adenoviral overexpression techniques and Btk-deficient
with actinomycin D (Figure 5C). XLA cells,14 and have the advantage of reducing potential developmen-
tal compensation between Tec kinases. Recent studies have questioned a
Increased Bmx levels regulate mRNA stability of TNF and IL-6 role for Btk in TLR-induced TNF production and p38 MAPK activation
through the 3ⴕUTR in human XLA cells. Studies by Perez de Diego et al showed that the
proportion of cells expressing phosphorylated p38 MAPK and intracel-
To examine the mechanism of this mRNA stabilization, we used
reporter gene constructs for TNF and IL-6 that contained the lular TNF and IL-6 as assessed by fluorescence-activated cell sorter
5⬘ promoter with and without the 3⬘UTRs as described previously14 analyses did not differ between XLA and controls.20 However, this is not
(Figure 6A). Bmx overexpression only increased reporter gene activity in conflict with our previous findings because XLA cells express
of the reporter constructs that contained the 3⬘UTR (Figure 6B). reduced levels of TNF and also show low levels of p38 MAPK
phosphorylation in response to LPS,14,17 indicating no change in the
proportion of cells producing TNF downstream of activated p38
MAPK, but a change in quantity of TNF production from the cell
Discussion
population. Sochorova et al showed differential levels of TLR-induced
This study has produced several key observations: We have TNF and IL-6 production by XLA-derived dendritic cells compared
confirmed that Btk regulates TLR-induced TNF, but not IL-6 with controls.21 This discrepancy could be the result of differences in
production, in primary human macrophages after LPS stimulation. TLR signaling between different cell types. We have previously shown
Subsequently, we have identified a novel role for Bmx in the that only undifferentiated XLA PBMCs show impaired TNF responses
regulation of TLR4-induced inflammatory cytokine production. after TLR stimulation.14 It is therefore possible that, like M-CSF
Further investigations into the mechanisms involved show, for the differentiated XLA PBMCs, XLA dendritic cells are able to compensate
first time, that Bmx overexpression results in increased TNF for the lack of functional Btk by increased expression of related kinases.
mRNA stabilization via a p38 MAPK-dependent mechanisms Contrasting to its family members Btk and Tec, Bmx protein
targeting the 3⬘UTR of the TNF gene. Moreover, our data clearly expression levels were not altered by M-CSF–driven differentia-
indicate a novel, thus far undefined, mechanism by which Bmx tion. In addition, we have shown here, for the first time, that Bmx is
induces increased IL-6 production through targeting of the 3⬘UTR activated by LPS stimulation in human macrophages and that
and mRNA stabilization. Crucially, increased IL-6 expression overexpression of this kinase leads to increased TNF and IL-6
downstream of Bmx does not involve changes in NF-B activity or production. This indicates overlapping roles for Btk and Bmx in the
require activation of the p38 MAPK signaling pathway. regulation of TNF, and a unique role for Bmx in the regulation of
Our observations using targeted knockdown of Btk through RNAi in IL-6 in human macrophages. We have previously shown that
human macrophages showing decreased TNF, but not IL-6, production undifferentiated and M-CSF-differentiated XLA PBMC produce
in response to LPS further validated our previous findings obtained normal levels of IL-6 after TLR4 engagement,17 which can now beFrom www.bloodjournal.org by guest on September 8, 2015. For personal use only.
BLOOD, 15 FEBRUARY 2008 䡠 VOLUME 111, NUMBER 4 Bmx REGULATES IL-6 PRODUCTION IN MACROPHAGES 1787
LFM-A13 as Btk inhibitor need to be reviewed in light of recent
findings that this inhibitor is not specific for Btk33,34 and was shown to be
toxic at high concentrations in our hands.
Concurring with previous findings by us and others showing
various degrees of compensation between Tec kinases,14,35,36 we
have now shown that Bmx shows overlapping functions with Btk in
targeting the 3⬘UTR of TNF to increase mRNA stabilization in a
p38 MAPK-dependent manner. LPS-induced IL-6 production in
human macrophages is not regulated by any of the MAP kinases, as
shown by inhibitor studies. Unexpectedly, further investigations
into the mechanisms involved in Bmx-dependent regulation of IL-6
revealed that Bmx targets the 3⬘UTR of the IL-6 gene to increase
mRNA stabilization via an as yet undefined, p38 MAPK-
independent pathway. It would have been satisfying to support our
data from Bmx overexpression with studies on Bmx-deficient cells.
However, no human phenotype equivalent to XLA is known for
Bmx, and knockdown of Bmx expression in macrophages using
siRNA oligonucleotides has thus far not been possible. We have
achieved knockdown of Bmx mRNA, but not protein, which might
indicate that the half-life of Bmx is too long for RNAi to have any
effect on constitutive protein levels. Furthermore, we have shown
that the Bmx kinase domain is required for LPS-induced super-
Figure 6. Bmx targets the 3ⴕUTRs of TNF and IL-6 after LPS stimulation.
induction of TNF and IL-6 using adenoviruses expressing
(A) Schematic representation of the human IL-6 5⬘ promoter-luciferase-3⬘UTR
(AdIL-6 5⬘/3⬘) and IL-6 5⬘ promoter-luciferase (AdIL-6 5⬘only) adenoviral constructs. kinase dead (K445E) and kinase deleted (⌬417-675) versions
(B) Macrophages were left uninfected or infected with Ad0 and AdBmx before a of Bmx (Figure S2, available on the Blood website; see the
second round of infections with AdTNF 5⬘/3⬘, AdTNF5⬘, AdIL-6 5⬘/3⬘, or AdIL-6
Supplemental Materials link at the top of the online article).
5⬘ luciferase reporter gene constructs and stimulated with LPS (10 ng/mL) for
4 hours. Relative luciferase units (RLU) for uninfected, Ad0-infected, and AdBmx- However, these mutants do not act as dominant negatives, as has
infected cells in the absence or presence of TNF reporter viruses (top panel) or IL-6 been observed by others.37,38
reporter viruses (bottom panel) after 4 hours of LPS stimulation are shown as mean
plus or minus SEM normalized to controls for 4 separate donors. Statistical
An increasing body of evidence from the laboratory and the
significance was assessed by Student t test (*P ⬍ .05; **P ⬍ .01). clinic indicates that different inflammatory pathologies involve
different cytokines and growth factors and that therapies need to be
explained by the presence of Bmx in these cells. The fact that Bmx closely tailored toward any particular type of pathologic inflamma-
may also have an impact on TNF expression would explain why, tion. We have shown in this study that 2 major pro-inflammatory
even in XLA cells, there is still some cytokine production. These cytokines, TNF and IL-6, are differentially regulated by Bmx
findings also explain why Btk siRNA knockdown does not result in a through downstream targeting of parallel, but distinct, pathways
dose-dependent decrease in TNF production, as Bmx could account for leading to increased mRNA stability of both cytokines. With the
some of the TNF production observed in response to LPS. clinical focus in anticytokine therapies shifting toward a broader
Our findings for Bmx are important for our current understanding of range of targets for different pathologies, it is important to
the inflammatory response because they highlight distinct differences in understand which precise mechanisms are involved in the produc-
the regulation of TNF and IL-6 in response to TLR stimulation. tion of different cytokines, and how this might differ depending on
Although LPS-induced IL-6 production is sensitive to inhibition of
cell type and trigger, as this might vary depending on the disease
NF-B activation, signaling downstream of Bmx leading to increased
and affected tissues.
IL-6 production does not involve changes in NF-B activity, as assessed
by inhibitor studies, luciferase reporter gene assays, and EMSA. These
findings are supported by the fact that we did not observe any
involvement of Btk signaling in NF-B signaling in primary human
macrophages,14 suggesting that these closely related kinases both signal
Acknowledgments
independently of NF-B in this model system. The apparent lack of
Bmx WT, Bmx HA, and Bmx kinase dead (KD) constructs and
involvement of NF-B downstream is further supported by our studies
anti-Bmx antibody were generously provided by Dr Michael
using the 5⬘ IL-6 promoter, which contains the IL-6 NF-B site and also
Tomlinson (Division of Medical Sciences, The Medical School,
showed no response to Bmx overexpression. However, we cannot
University of Birmingham, Birmingham, United Kingdom). Dr
exclude any possible effects of Bmx on IL-6 transcription, such as the
phosphorylation of histones; this is the subject of future investigations. David Webster led the collaboration with the Royal Free Medical
Differing reports regarding the role of Btk signaling in NF-B activation School, Department of Immunology. XLA blood samples were
in response to TLR engagement have been published in the literature collected by Dr Lynett Danks. Volunteer venesectionists and
and need to be analyzed in the context of the model systems used. control blood donors were employees at the Kennedy Institute of
Studies implicating a role for Btk in the regulation of NF-B, and not Rheumatology. Dr Andy Clark and Dr Jeremy Turner provided
p38 MAPK, activation were conducted in nonmyeloid cell lines15 and critical feedback.
murine models,16,22 and conflicting findings might thus be the result of This work was supported by grants from the Medical Research
differences between species, as well as myeloid and nonmyeloid cell Council United Kingdom, Arthritis Research Campaign United
systems. In addition, studies conducted using high concentrations of Kingdom, and Kennedy Institute of Rheumatology Trustees.From www.bloodjournal.org by guest on September 8, 2015. For personal use only.
1788 PALMER et al BLOOD, 15 FEBRUARY 2008 䡠 VOLUME 111, NUMBER 4
and B.M.J.F. designed research and wrote the manuscript.
Authorship Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Contribution: C.D.P. designed and performed research, analyzed Correspondence: Brian M. J. Foxwell, Kennedy Institute of
data, and wrote the manuscript; B.E.M. and J.P.M. helped with Rheumatology, Imperial College London, 1 Aspenlea Road, Lon-
performing research; S.W. provided XLA blood samples; N.J.H. don, W6 8LH, United Kingdom; e-mail: b.foxwell@imperial.ac.uk.
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2008 111: 1781-1788
doi:10.1182/blood-2007-07-102343 originally published
online November 19, 2007
Bmx tyrosine kinase regulates TLR4-induced IL-6 production in human
macrophages independently of p38 MAPK and NF κB activity
Christine D. Palmer, Brenda E. Mutch, Sarita Workman, John P. McDaid, Nicole J. Horwood and
Brian M. J. Foxwell
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