Involvement of hypoxia-inducible factor-1 in the inflammatory responses of human LAD2 mast cells and basophils.

Abstract

We recently showed that hypoxia-inducible factor 1 (HIF-1) plays a crucial role in the pro-allergic functions of human basophils by transcriptional control of energy metabolism via glycolysis as well as directly triggering expression of the angiogenic cytokine vascular endothelium growth factor (VEGF). Here, we investigated HIF-1 involvement in controlling the synthesis of angiogenic and inflammatory cytokines from various human effector cells stimulated by IgE-dependent or innate immune triggers. Purified primary human basophils, LAD2 human mast cells and THP-1 human myeloid cells were used for investigations of FcεRI and Toll-like receptor (TLR) ligand-induced responses. In contrast to basophils, LAD2 mast cells expressed background levels of HIF-1α, which was largely independent of the effects of stem cell factor (SCF). Both mast cells and basophils expressed TLR2 and 4, albeit weakly compared to THP-1 cells. Cytokine production in mast cells following TLR ligand stimulation was markedly reduced by HIF-1α knockdown in LAD2 mast cells. In contrast, although HIF-1 is involved in IgE-mediated IL-4 secretion from basophils, it is not clearly induced by peptidoglycan (PGN). HIF-1α accumulation is critical for sustaining human allergic effector cell survival and function. This transcription complex facilitates generation of both pro-angiogenic and inflammatory cytokines in mast cells but has a differential role in basophil stimulation comparing IgE-dependent triggering with innate immune stimuli.

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Involvement of Hypoxia-Inducible Factor-1 in the
Inflammatory Responses of Human LAD2 Mast Cells and
Basophils
Vadim V. Sumbayev.*, Inna Yasinska., Abraham E. Oniku, Claire L. Streatfield, Bernhard F. Gibbs*
Medway School of Pharmacy, University of Kent, Chatham Maritime, United Kingdom
Abstract
We recently showed that hypoxia-inducible factor 1 (HIF-1) plays a crucial role in the pro-allergic functions of human
basophils by transcriptional control of energy metabolism via glycolysis as well as directly triggering expression of the
angiogenic cytokine vascular endothelium growth factor (VEGF). Here, we investigated HIF-1 involvement in controlling the
synthesis of angiogenic and inflammatory cytokines from various human effector cells stimulated by IgE-dependent or
innate immune triggers. Purified primary human basophils, LAD2 human mast cells and THP-1 human myeloid cells were
used for investigations of FceRI and Toll-like receptor (TLR) ligand-induced responses. In contrast to basophils, LAD2 mast
cells expressed background levels of HIF-1a, which was largely independent of the effects of stem cell factor (SCF). Both
mast cells and basophils expressed TLR2 and 4, albeit weakly compared to THP-1 cells. Cytokine production in mast cells
following TLR ligand stimulation was markedly reduced by HIF-1a knockdown in LAD2 mast cells. In contrast, although HIF-
1 is involved in IgE-mediated IL-4 secretion from basophils, it is not clearly induced by peptidoglycan (PGN). HIF-1a
accumulation is critical for sustaining human allergic effector cell survival and function. This transcription complex facilitates
generation of both pro-angiogenic and inflammatory cytokines in mast cells but has a differential role in basophil
stimulation comparing IgE-dependent triggering with innate immune stimuli.
Citation: Sumbayev VV, Yasinska I, Oniku AE, Streatfield CL, Gibbs BF (2012) Involvement of Hypoxia-Inducible Factor-1 in the Inflammatory Responses of Human
LAD2 Mast Cells and Basophils. PLoS ONE 7(3): e34259. doi:10.1371/journal.pone.0034259
Editor: Michael B. Fessler, National Institute of Environmental Health Sciences, United States of America
Received January 13, 2012; Accepted February 24, 2012; Published March 28, 2012
Copyright: ? 2012 Sumbayev et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Work was supported by the Asthma UK Grant (grant number 10/065 - to BFG and VS). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: B.F.Gibbs@kent.ac.uk (BFG); V.Sumbayev@kent.ac.uk (VVS)
. These authors contributed equally to this work.
Introduction
Human allergic and inflammatory reactions are associated with
the activation of effector cells in a ligand-receptor dependent
manner [1,2]. Mast cells and basophils are key effectors of allergic
inflammation whereas other myeloid cells, such as monocytes/
macrophages and neutrophils, mediate pathogen-induced host
innate immune reactions [3]. Allergic inflammation is governed by
high-affinity IgE receptor (FceRI)-IgE-allergen complexes leading
to histamine release and production of pro-inflammatory cyto-
kines/eicosanoids [1,2]. Conversely, pathogen-induced inflamma-
tory responses are triggered via pathogen-associated molecular
patterns detected by pro-inflammatory Toll-like receptors (TLRs)
that induce the expression of inflammatory cytokines in effector
cells.
All the above inflammatory responses require effector cells to
adapt to stress induced by pro-inflammatory stimulation. The
adaptation process is controlled by a number of mechanisms,
where the crucial one is activation of the hypoxia-inducible factor
1 (HIF-1) transcription complex [4–6]. This complex plays a
pivotal role in cellular adaptation to low oxygen availability and to
inflammatory stress [4]. HIF-1 is a heterodimeric transcription
complex containing the constitutive beta and an inducible alpha
subunit, accumulation of which determines HIF-1 transcriptional
activity [4]. HIF-1 has a number of target genes (over 40) that
control angiogenesis, cell adhesion and glycolysis [4,5].
We recently reported that the pro-allergic (IgE-mediated)
responses of primary human basophils, as well as their capacity
to generate the angiogenic cytokine VEGF, involve HIF-1
activation [6]. Different kinds of inflammatory stress, such as
pathogen-associated molecular pattern-induced TLR-mediated
triggering, were also found to activate HIF-1 in THP-1 monocytes
[7–8]. In all cases HIF-1 directly controlled VEGF expression on
the transcriptional level and facilitated pro-inflammatory cytokine
expressions by upregulating glycolysis (thus controlling intracellu-
lar ATP levels and preventing their depletion). However, HIF-1
accumulation in THP-1 cells and basophils was governed by
differential intracellular signalling mechanisms [6–8].
Mast cells and basophils not only express FceRI receptors but
also several TLRs (in particular, TLR 2 and TLR 4 that recognise
molecular patterns shared mostly by bacterial pathogens [9–13]).
Thus, in case of an infection in an asthmatic airway mast cells and
basophils could be stimulated by pathogen-associated molecular
patterns through TLRs and by IgE-dependent mechanisms [9–
13]. Monocytes/macrophages, on the other hand, which are
usually regarded as key effectors of host innate immune defences,
also express FceRII (CD23) and thereby can participate in allergic
responses too [14]. Therefore, these cell types seem capable of
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contributing to both innate immune and allergic responses.
However, it is unclear whether these inflammatory receptors
could simultaneously react to both IgE-dependent stimuli and
TLR ligands leading to dual inflammatory stress, thus affecting -
for example by potentiation- respective types of inflammatory
reactions. It is also unclear whether HIF-1 accumulation and
modulation of pro-inflammatory cytokine generation differs
between human basophils and mast cells. Given the above, in
the present study we addressed the role of HIF-1 in human mast
cell responses to IgE- and TLR-mediated triggering in comparison
to human basophils and THP-1 monocytes.
Materials and Methods
Materials
Anti-human IgE, RPMI-1640 medium, foetal calf serum and
supplements, DOTAP transfection reagent, enhanced avian HS
RT-PCR kit, GenEluteTMmammalian total RNA miniprep kit,
LPS, PGN, Pam3Cys and HIF-1a-specific siRNA were purchased
from Sigma (Suffolk, UK). MaxisorpTMmicrotitre plates were
obtained from Nunc (Roskilde, Denmark). ELISA-based assay kits
for detection of VEGF, TNF-a and IL-6, as well as a caspase 3
assay kit, were bought from R&D Systems (Abingdon, UK). IL-4
ELISA kits were purchased from Biolegend (Cambridge BioSci-
ence Ltd, UK). Mouse monoclonal antibody to HIF-1a, mouse
monoclonal antibody to b-actin as well as rabbit polyclonal HRP-
labelled antibody to mouse IgG were from Abcam (Cambridge,
UK). Human polyclonal IgE was purchased from Amsbio
(Abingdon, UK). Stem-Pro-34 serum-free media and stem cell
factor (SCF) were obtained from Invitrogen (Paisley, UK).
Quantitative real-time PCR kit and real-time PCR plates were
bought from Roche (Burgess Hill, UK). All other chemicals were
of the highest grade of purity and commercially available (obtained
from Sigma (Suffolk, UK)).
LAD2 mast cells
LAD2 mast cells were kindly provided by A. Kirshenbaum and
D. Metcalfe (NIH, USA) [15]. Cells were cultured in the Stem-
Pro-34 serum-free media in the presence of 100 ng/ml SCF.
Primary human basophils
Human basophils were obtained from buffy coats acquired from
healthy blood donors undergoing routine blood donation following
ethical approval. This research was approved by the NHS-REC
(ref. number: 07/Q1206/3), Blood samples were purchased from
the UK National Health Service (NHS) Blood and Transplant
(NHSBT) service and were collected in accordance with their
internal regulations. Basophils were purified by Ficoll density
centrifugation followed by negative selection using magnetic cell
sorting [16]. Basophils (9462% pure, as determined by alcian blue
staining with viability of .95%) were incubated for 15 min at
37uC in HEPES-buffered Tyrode’s solution and treated as
indicated. Following stimulation, histamine release analysis and
cell lysis was performed as previously described [6].
THP-1 human myeloid cells and HEK293 cells
THP-1 human leukaemia monocytic macrophages were
purchased from the European collection of Cell Cultures (Salis-
bury, UK). Cells were grown in RPMI 1640 media supplemented
with 10% foetal calf serum, penicillin (50 IU/ml) and streptomy-
cin sulphate (50 mg/ml). HEK293 cells were kept at the same
conditions.
Transfer of HIF-1a siRNA to LAD2 mast cells
HIF-1a-specific siRNA, together with its mutated form (which
was used as an inactive control), was obtained from Sigma.
Transfection into LAD2 cells was performed using DOTAP
reagent [8,17] according to the manufacturer’s protocol. As we
previously reported [18], mutated siRNA did not impact the
investigated processes (data not shown), confirming that the effects
observed were caused by knocking down specific HIF-1a when
respective specific siRNAs were used. siRNA was transfected into
LAD2 cells using DOTAP transfection reagent according to the
manufacturer’s protocol. The efficiency of HIF-1a knockdown was
4767%.
Western blot analysis
HIF-1a, TLR2 and TLR4 were determined by Western blot
analysis and compared to b-actin expressions to assess equal
loading, as described in our previous publication [8]. Li-Cor
(Lincoln, Nebraska USA) goat secondary antibodies conjugated
with fluorescent dyes for 60 min were employed according to the
manufacturer’s protocol and proteins visualized using a Li-Cor
Odyssey imaging system. The Western blot data were subjected to
quantitative analysis using Odyssey software and values were
normalised against respective actin bands.
Measurement of HIF-1a and VEGF by quantitative real-
time reverse transcription PCR (qRT-PCR)
Total RNA was isolated using GenEluteTMmammalian total
RNA miniprep kit, followed by HIF-1a mRNA reverse transcrip-
tase–polymerase chain reaction (RT-PCR) performed in accor-
dance with the manufacturer’s protocol. Then quantitative real-
time PCR was performed. Primer selection was as follows: HIF-
1a, 59-CTCAAAGTCGGACAGCCTCA-39, 59-CCCTGCAG-
TAGGTTTCTGCT-39 VEGF, 59-GTATAAGTCCTGGAGC-
GT-39, 59-CTCGGAGGGAGTCCCAAA-39; actin, 59-TGACG-
GGGTCACCCACACTGTGCCCATCTA-39,
CATT-TGCGGTCGACGATGGAGGG-39 [8]. Reactions were
performed using a LightCyclerH 480 real-time PCR system and
respective SYBR Green I Master kit (Roche, Burgess Hill, UK).
Analysis was performed according to the manufacturer’s protocol.
Values representing VEGF and HIF-1a mRNA levels were
normalised against those of actin.
59-CTAGAAG-
Measurement of TNF-a, VEGF, IL-4 and IL-6 production
Production of TNF-a, VEGF, IL-4 and IL-6 by the cells was
analysed by ELISA according to the manufacturer’s protocols.
Histamine release
Mast cell and basophil histamine releases were assessed using
spectrofluorometic autoanalysis as previously described [6,19].
Percentage histamine releases were calculated from the total
histamine content in the sum of lysed cell pellet and supernatant
vials [6,19].
Detection of intracellular ATP
This was analysed using a luminometric kit (Sigma) according to
the manufacturer’s protocol.
Measurement of Caspase 3 activity
The activity of caspase 3 was assayed by a colorimetric method
based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-
Val-Asp-p-nitroanilide (Ac-DEVD-pNA) and carried out accord-
ing to the manufacturer’s protocol.
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Figure 1. LAD2 human mast cells and primary human basophils express detectable amounts of TLRs 2 and 4. LAD2 cells, primary
human basophils (PHB) and THP-1 cells (positive control) were subjected to in-cell TLR2 (A) and TLR4 (B) assays (upper panel). TLRs 2 (A) and 4 (B)
were also detected in the cell lysates (lower panel). Quantitative data are mean values+S.D. of at least three individual experiments. * indicates
p,0.01 vs. control. All Western blot data shown are from one representative experiment out of three that gave similar results.
doi:10.1371/journal.pone.0034259.g001
Figure 2. PGN and LPS induce HIF-1a accumulation in THP-1 but not in LAD2 cells. THP-1, LAD2 and HEK293 (negative control – lacking
TLR2/4 expression) cells were exposed for 4 h to 1 mg/ml PGN or LPS. HIF-1a accumulation was then analysed. Quantitative data are mean
values+S.D. of at least three individual experiments. * indicates p,0.01 vs. control. All Western blot data shown are from one representative
experiment out of three that gave similar results.
doi:10.1371/journal.pone.0034259.g002
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MTS cell viability assay
Cell viability was analysed using the Promega (Southampton,
UK)3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assay kit
according to the manufacturer’s protocol.
In-cell TLR2 and TLR4 assays
This was performed as recently described [20,21]. Briefly, cells
were centrifuged for 5 min at 1200 rpm, washed with respective
media and exposed to 2 mg/ml anti-TLR2 or anti-TLR4 antibody
for 2 h. This was followed by centrifugation for 5 min at
1200 rpm and washing with media. Cells were then incubated
for 2 h with fluorescent dye-labelled Li-Cor (Cambridge, UK)
secondary antibodies. Following centrifugation/washing, the cells
were transferred into wells of a 96-well plate which was scanned by
the Li-Cor Odyssey imaging system. Results were quantified using
Odyssey software.
Statistical analysis
Each experiment was performed at least three times and
statistical analysis was conducted using two-tailed Student’s t
test. Statistical probabilities (p) were expressed as *, where
p,0.01.
Results
LAD2 mast cells and primary human basophils express
TLRs 2 and 4
Human allergic effector cells (mast cells and basophils) are
known to produce detectable levels of TLRs 2 and 4 mRNA
[9,10]. Translation of these mRNAs into functional plasma
membrane-associated receptors is, however, still questionable.
We therefore studied whether LAD2 human mast cells and
primary human basophils express TLR2/4 proteins. To analyse
this we used Western blot analysis and in-cell TLR2/4 assays.
THP-1 human myeloid cells were used as a positive control as they
are known to express functional forms of both TLRs [7,22]. We
found that both mast cells and basophils expressed detectable
amounts of TLRs 2 and 4, although these expression levels were
significantly lower compared to those observed in THP-1 cells
(Figure 1).
LAD2 mast cells accumulate HIF-1a protein which is not
upregulated in a TLR2/4-dependent manner
Recent evidence has demonstrated involvement of the HIF-1
transcription complex in TLR-mediated inflammatory reactions
[5–8]. We therefore asked whether this case is applicable to LAD2
Figure 3. PGN and LPS do not potentiate IgE-induced responses of LAD2 mast cells. LAD2 cells were sensitised with 100 ng/ml IgE for
24 h followed by 4 h exposure to 1 mg/ml PGN or LPS. Thereafter, some samples (including TLR ligand treated and untreated) were exposed to
0.1 mg/ml anti-IgE for 2 h. HIF-1a accumulation, VEGF mRNA expression/VEGF release, histamine and TNF-a release as well as intracellular ATP levels
were analysed as described in Materials and methods. Quantitative data are mean values+S.D. of at least three individual experiments. * indicates
p,0.01 vs. control. All Western blot data are from one experiment representative of three that gave similar results.
doi:10.1371/journal.pone.0034259.g003
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human mast cells. In THP-1 human myeloid cells we found that
4 h of exposure to 1 mg/ml PGN or LPS led to a significant
increase in HIF-1a accumulation (Figure 2). Similar experiments
performed with LAD2 mast cells demonstrated HIF-1a accumu-
lation in non-treated cells. Exposure to TLR2/4 ligands did not
change the intracellular levels of stabilised HIF-1a protein
(Figure 2). HEK293 cells were used as a negative control (they
do not express TLRs at all and we have not detected any
expression of TLRs 2 and 4 in these cells by in-cell assay and
Western blot – data not shown) and did not respond to stimulation
with TLR2/4 ligands (Figure 2).
PGN/Pam3Cys and LPS do not potentiate IgE-mediated
pro-allergic responses of LAD2 human mast cells
We investigated whether TLRs 2 and 4 induce pro-inflamma-
tory responses in LAD2 human mast cells. We were also
interested whether TLR2/4 ligands potentiate IgE-mediated
pro-allergic responses in LAD2 mast cells. Cells were therefore
sensitised with 100 ng/ml IgE for 24 h followed by 4 h exposure
to 1 mg/ml PGN or LPS (some of the samples were left
untreated). Some of the cell samples (including TLR ligand
treated and untreated) were then exposed to 0.1 mg/ml anti-IgE
for 2 h. We observed that neither TLR2/4 ligands nor anti-IgE
alone induced further upregulation of constitutive HIF-1a
accumulation. Co-stimulation with PGN or LPS and anti-IgE
did not lead to any HIF-1a upregulation either (Figure 3). The
Figure 4. Pam3Cys does not potentiate Anti-IgE response of
LAD2 human mast cells. LAD2 cells were sensitised with 100 ng/ml
IgE for 24 h followed by 4 h exposure to 1 mg/ml Pam3Cys. Thereafter,
some samples (including TLR ligand treated and untreated) were
exposed to 0.1 mg/ml anti-IgE for 2 h. Histamine, VEGF and TNF-a
release was analysed as described in Materials and methods.
Quantitative data are mean values+S.D. of at least three individual
experiments. * indicates p,0.01 vs. control.
doi:10.1371/journal.pone.0034259.g004
Figure 5. Anti-IgE dose-dependent responses of LAD2 human
mast cells. LAD2 human mast cells were sensitised with 100 ng/ml IgE
for 24 h followed by 2 h of exposure to increasing concentrations of
anti-IgE. HIF-1a accumulation, VEGF mRNA expression/VEGF release,
histamine and TNF-a release as well as intracellular ATP levels were
analysed as described in Materials and methods. Quantitative data are
mean values 6 S.D. of at least three individual experiments. * indicates
p,0.01 vs. control. All Western blot data are from one experiment
representative of three that gave similar results.
doi:10.1371/journal.pone.0034259.g005
HIF-1 in Mast Cells and Basophils
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