Ann. occup. Hyg., Vol. 46, Supplement 1, pp. 436–439, 2002
© 2002 British Occupational Hygiene Society
Published by Oxford University Press
DOI: 10.1093/annhyg/mef711
PM10-mediated IL-8 Release from Epithelial Cells
Involves Histone Acetylation
P. S. GILMOUR1*, I. RAHMAN1, V. STONE2, K. DONALDSON1,2 and
W. MacNEE1
1Edinburgh
Lung and the Environment Group Initiative (ELEGI)/Colt Laboratory, The University of
Edinburgh, Wilkie Building, Department of Medicine and Radiological Sciences, Medical School, Teviot
Place, Edinburgh EH8 9AG; 2The School of Life Sciences, Napier University, 10 Colinton Road,
Edinburgh EH10 5DT, UK
Increases in the levels of environmental particulate matter (PM10) in the air are associated with
a variety of adverse health effects, particularly in susceptible patients with chronic lung and
cardiovascular diseases. The adverse effects in the lungs are probably caused by oxidative stress
leading to lung inflammation. The expression of many genes, including those encoding proinflammatory mediators, involves the remodelling of the chromatin structure provided by
histone proteins. Chromatin is tightly coiled around histone proteins and therefore the access
of transcription factors to the transcriptional machinery is inhibited. Histone acetylation causes
the unwinding of the chromatin structure, thereby allowing transcription factor access to
promoter sites, whereas histone deacetylation has the opposite effect of winding chromatin and
inhibiting transcription. Nuclear histone acetylation is reversible and is regulated by a group of
histone acetyltransferases (HATs), which promote acetylation, and histone deacetylases
(HDACs), which promote deacetylation. There are several co-activators, transcription factors
and nuclear proteins that have HAT activity. The aim of this study was to determine whether
the PM10-mediated mRNA expression and release of interleukin-8 (IL-8) from alveolar airspace
epithelial cells is associated with histone acetylation and oxidative stress. We studied the effects
of PM10 and an HDAC inhibitor (trichostatin-A, TSA) on the release of IL-8 by enzyme-linked
immunosorbent assay, and the acetylation of histone 4 (H4) was assessed by immunocytochemistry in human alveolar type II cells. PM10 and H2O2 increased IL-8 protein release from A549
cells after 20 h treatment, and this was enhanced by HDAC inhibition (TSA co-treatment).
PM10 and H2O2 treatment also increased the HAT activity. PM10 enhanced H4 acetylation,
which was mediated by oxidative stress, as shown by thiol antioxidant inhibition. The PM10and TSA-mediated increases in IL-8 and histone acetylation were associated with increases in
nuclear factor-κB activation. These data suggest that the remodelling of chromatin by histone
acetylation plays a role in the PM10-mediated pro-inflammatory responses in the lungs. This
PM10 response is mediated by oxidative stress.
Keywords: PM10; histone acetylation; IL-8; A549
et al., 2000). PM10 particles have been shown to produce free radicals and are likely to exert an oxidative
stress upon lung cells. One consequence of oxidative
stress is the activation of transcription factors, such as
NF-κB and activator protein-1 (AP-1) (Gilmour et
al., 1996). PM10 also enhances interleukin-8 (IL-8)
gene expression and protein release from lung epithelial cells by a mechanism involving oxidative
stress and transcription factor activation (Jimenez et
al., 2000; Gilmour et al., 2001).
The transcription of many genes is known to correlate
with levels of acetylated nuclear histone proteins
INTRODUCTION
Increases in the concentration of environmental particles (PM10) are associated with a variety of respiratory adverse health effects, including increased
hospital admissions for exacerbations of asthma and
chronic obstructive pulmonary disease (COPD), and
increased deaths due to respiratory and cardiovascular causes (Dockery and Pope 1996; MacNee
*Author to whom correspondence should be addressed.
e-mail: [email protected]
436
PM10-mediated IL-8 release from epithelial cells
(Grunstein, 1997). Histone acetylation/deacetylation
plays a critical role in the remodelling of chromatin
and therefore gene expression (Youn et al., 2000).
This is because DNA is packaged in a tight coil
around a core of four histone proteins, H2A, H2B, H3
and H4, which make up the nucleosome (Fig. 1). The
acetylation of the histone core of the chromatin
promotes unwinding of chromatin and the access of
transcription factors and co-activators to target gene
promoter sites, thus initiating transcription. Conversely,
the deacetylation of the histone core prevents the
chromatin unwinding, which inhibits access of
transcription factors to the target sites on DNA and
therefore inhibits gene transcription. Acetylation is
mediated by compounds with histone acetyltransferase (HAT) activity, and a family of histone
deacetylases (HDACs) inhibit acetylation (Kuo and
Allis, 1998). There are many nuclear associated
proteins which possess intrinsic HAT activity, and
several HDAC enzymes (Kuo and Allis, 1998).
Cigarette smoke-mediated oxidative stress has
been associated with changes in histone acetylation
status in lung phagocytic cells (Ito et al., 2001). This
led us to hypothesize that the pro-inflammatory effects
of PM10-mediated oxidative stress may involve alterations in the histone acetylation/deacetylation balance,
facilitating gene transcription. Modification of histone
acetylation status may therefore play a role in the
expression and release of inflammatory mediators.
We therefore studied the effects of PM10 and the
HDAC inhibitor trichostatin-A (TSA) on release of
the pro-inflammatory cytokine IL-8 from alveolar
epithelial (A549) cells.
437
MATERIALS AND METHODS
Cell culture
A549 (a human lung alveolar type II cell line) cells
were maintained in culture as described previously
(Gilmour et al., 2001).
Cell treatments
For cell treatments, the medium on the 80%
confluent monolayers was replaced with serum-free
medium, the cells incubated for 24 h and treatments
added for relevant time in serum-free medium.
Particulate matter of which 50% was <10 µm diameter (PM10) was obtained, quantified and used as
previously described (Gilmour et al., 2001). Cells
were treated with 100 ng/ml TSA, 100 µg/ml PM10,
100 µM H2O2 and 5 mM N-acetyl-L-cysteine (NAC)
for 20 h.
HAT activity and histone extraction
Cells were incubated with 0.05 mCi [3H]acetic acid
for 10 min followed by co-incubation with test agents
for 2 h. The [3H]acetic acid is the substrate for the
acetylation reaction and becomes incorporated into
acetylated histones. Nuclei were then extracted as
described previously (Gilmour et al., 2001). Acidsoluble histones were purified and acetone precipitated by the method described by Ito et al. (2000).
They were then separated by sodium dodecylsulphate–polyacrylamide gel electrophoresis, and the
H4 protein was identified by comparison with a
standard H4 peptide and cut out of the gel. The HAT
activity (acetylation of H4) was determined by liquid
Fig. 1. Regulation of chromatin configuration by histone acetylation/deacetylation. DNA helix is tightly coiled around histone
proteins. Acetylation of these histones by HAT activity unwinds the DNA coil, promoting access of transcription factors and coactivators to target gene promoter sites and enhancing gene transcription. HDAC activity represses the acetylation of histones and
therefore inhibits DNA unwinding and transcription. The HDAC activity is prevented by TSA treatment.
438
P. S. Gilmour et al.
scintillation counting of H4-incorporated [3H]acetic
acid, producing an activity value of disintegrations
per minute (d.p.m.) per µg of histone protein.
Immunocytochemistry of acetylated histone protein
H4
Cells grown on coverslips after treatment were
fixed in ice-cold methanol for 10 min before being
blocked with 8% BSA. The cells were washed and
incubated with goat polyclonal anti-acetylated
human H4 antibody as the primary antibody for 1 h
at room temperature (Upstate Biotechnology, Lake
Placid, NY). The cells were incubated with goat antirabbit IgG Alexa red as a secondary antibody (Molecular Probes, Cambridge, UK) and finally stained with
Hoechst dye. Images of cellular immunofluorescence
were acquired using a high-resolution fluorescence
microscope (Zeiss) with a digital camera (CoolSnap)
attached to a G3 Apple MacIntosh computer, using
OpenLab software. Results were obtained as immunocytochemistry scores in which at least 300 cells were
counted and the percentage of acetylated cells (Alexa
Red positive) to total cell number (Hoescht positive)
was calculated.
IL-8 measurement
IL-8 release was determined as previously
described (Gilmour et al., 2001).
NF-κB activation
The nuclear activation of NF-κB was determined
by gel mobility shift analysis as previously described
(Gilmour et al., 2001).
Statistical analysis
One- or two-way analysis of variance was used to
determine the significance of treatment effects.
RESULTS
A549 cells treated with PM10 and H2O2 released
increased amounts of IL-8 after 20 h incubation
(Fig. 2). Inhibition of HDACs by TSA also increased
the release of IL-8 (Fig. 2). The addition of TSA to
the PM10 and H2O2 treatments augmented the IL-8
release mediated by these treatments alone (Fig. 2).
Inhibition of HDAC with TSA for 2 h increased the
HAT activity associated with H4 as determined by
the incorporation of [3H]acetic acid by 104%. Similarly, treatment with PM10 and H2O2 for 2 h increased
the HAT activity associated with H4 by 245% and
166%, respectively.
The presence of nuclei staining positive for
acetylated H4 was increased by TSA (189%) and
PM10 (261%) treatment for 20 h. The PM10-associated acetylation of H4 was shown to be mediated by
oxidative stress, as shown by the amelioration of
Fig. 2. IL-8 protein release following 20 h exposure to TSA,
H2O2, PM10, and H2O2 and PM10 with and without TSA
compared with untreated cells. Expressed as a percentage of
the untreated control. ***P < 0.001, **P < 0.01, *P < 0.05
compared with the untreated control.
Fig. 3. NF-κB activation following 20 h exposure to TSA and
PM10 compared with untreated cells.
acetylation (167% decrease in acetylated H4 positive
cells) by treatment with the thiol antioxidant NAC.
The activation of the transcription factor NF-κB
was enhanced by inhibition of HDAC with TSA and
by treatment with PM10 (Fig. 3).
DISCUSSION
We have shown that PM10-mediated IL-8 protein
release is associated with an increase in the presence
of acetylated H4 in the nuclei of A549 alveolar
epithelial cells. Furthermore, the inhibition of HDAC
enzymes by TSA also increased the levels of
acetylated H4, and enhanced the release of proinflammatory cytokine IL-8. This is the first study to
establish a link between the exposure to environmental particles, chromatin remodelling and proinflammatory gene transcription. The role of the
nucleosome remodelling in the control of gene transcription co-activator and transcription factor access
to the target promoter sites of genes is increasingly
viewed as vital for the transcriptional activation of
genes. The levels of histone acetylation have been
directly related to the levels of gene transcription
(Mizuguchi et al., 2001). Furthermore, histone acetylation has been reported to play a role in IL-8 and IL6 gene expression (Berghe et al., 1999, Wen and Wu,
2001). Our study supports the role of histone acetylation as a mechanism in inflammatory gene transcription. We have previously shown that NF-κB
PM10-mediated IL-8 release from epithelial cells
activation is an important feature of PM10-mediated
cytokine expression (Jimenez et al., 2000; Gilmour
et al., 2001). In this study we show that promotion
of histone acetylation by TSA enhances NF-κB activation. A role for histone acetylation and NF-κB
activation has been reported for the transcription of
another pro-inflammatory cytokine, IL-6 (Berghe et
al., 1999). NF-κB is not only activated by this mechanism but is also associated with co-activators, such
as CBP/p300, which themselves promote the acetylation of histones (Berghe et al., 1999).
Histone acetylation has been reported in response
to cytokines (IL-1, granulocyte colony-stimulating
factor) and cigarette smoke (Ito et al., 2001; Miyata
et al., 2001), stimuli that activate cells by signal
transduction and oxidative stress mechanisms. PM10
has been shown to exert oxidative stress (Gilmour et
al., 1996) and diesel particles, components of PM10,
can activate signal transduction in cells (Hashimoto
et al., 2000). As MAP kinase activation has been
associated with histone acetylation (Miyata et al.,
2001), this cell signalling pathway may play a role in
the histone-mediated pro-inflammatory effects seen
in this study. Furthermore, a role for both MAP
kinase activation and oxidative stress has been associated with changes in histone acetylation (Tikoo et al.,
2001). The PM10-induced increase in H4 acetylation
in this study was inhibited by the thiol antioxidant
NAC, suggesting that PM10 produces its effect via
oxidative stress. Oxidative stress causing depletion of
antioxidants (glutathione) can activate MAP kinase
pathways, specifically ERK and JNK, and by activation of these pathways these agents may regulate
histone acetylation. Similar events may follow PM10mediated oxidative stress. We show here that PM10
also has pro-inflammatory effects via oxidative stress
and histone acetylation, which provide a plausible
additional mechanism in the pro-inflammatory
effects of PM10.
CONCLUSIONS
PM10 and H2O2 increased levels of acetylated
histones and acetylated H4. This was associated with
the activation of NF-κB and the release of the proinflammatory cytokine IL-8. The HDAC inhibitor
TSA also increased IL-8 release. PM10 increased H4
acetylation by a mechanism involving oxidative stress,
as shown following co-incubation with NAC. These
results indicate a mechanism whereby environmental
particles can increase their pro-inflammatory activity
via an alteration in the balance between histone
acetylation and deacetylation in epithelial cells. This
indicates a mechanism for the pro-inflammatory
effects of PM10.
439
Acknowledgements—This work was supported by the British
Lung Foundation, the Medical Research Council (UK) and The
Colt Foundation. K.D. is the Transco British Lung Foundation
Fellow in Air Pollution and Respiratory Health.
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