Protective role of metallothionein in acute lung injury
induced by bacterial endotoxin
H Takano, K Inoue, R Yanagisawa, M Sato, A Shimada, T Morita, M Sawada, K Nakamura,
C Sanbongi, T Yoshikawa
See end of article for
Dr H Takano,
Team, National Institute for
16-2 Onogawa, Tsukuba
Received 3 March 2004
Accepted 19 July 2004
Thorax 2004;59:1057–1062. doi: 10.1136/thx.2004.024232
Background: Metallothionein (MT) is a protein that can be induced by inflammatory mediators and
participate in cytoprotection. However, its role in inflammation remains to be established. A study was
undertaken to determine whether intrinsic MT protects against acute inflammatory lung injury induced by
bacterial endotoxin in MT-I/II knock out (2/2) and wild type (WT) mice.
Methods: MT (2/2) and WT mice were given vehicle or lipopolysaccharide (LPS, 125 mg/kg)
intratracheally and the cellular profile of the bronchoalveolar lavage (BAL) fluid, pulmonary oedema, lung
histology, expression of proinflammatory molecules, and nuclear localisation of nuclear factor-kB (NF-kB)
in the lung were evaluated.
Results: MT (2/2) mice were more susceptible than WT mice to lung inflammation, especially to lung
oedema induced by intratracheal challenge with LPS. After LPS challenge, MT deficiency enhanced
vacuolar degeneration of pulmonary endothelial cells and type I alveolar epithelial cells and caused focal
loss of the basement membrane. LPS treatment caused no significant differences in the enhanced
expression of proinflammatory cytokines and chemokines nor in the activation of the NF-kB pathway in the
lung between the two genotypes. Lipid peroxide levels in the lungs were significantly higher in LPS treated
MT (2/2) mice than in LPS treated WT mice.
Conclusions: Endogenous MT protects against acute lung injury related to LPS. The effects are possibly
mediated by the enhancement of pulmonary endothelial and epithelial integrity, not by the inhibition of the
has been proposed that MT may have an important role in
homeostasis and detoxication of heavy metals.1It can react
also with free radicals and electrophils because of its high
sulfhydryl content,2 3and can serve as a sacrificial scavenger
for hydroxyl radicals in vitro4and also protects against free
radical induced DNA damage.5–7In addition, MT is induced
by heavy metals or oxidative stress producing chemicals,8and
exhibits cytoprotection against toxicity of heavy metals or
alkylating anticancer drugs3as well as against oxidative
stress related organ damage.9 10MT-I and MT-II genes are
constitutively expressed in the liver and are highly induced by
Proinflammatory cytokines, including tumour necrosis factor
(TNF)-a, interleukin (IL)-1, IL-6, and interferon-c, induce
hepatic MT gene expression in vivo. However, there are
conflicting reports about the role of MT in inflammatory
processes. In brief, MT plays a pivotal role in mediating the
harmful effects of TNF induced shock11but MT (2/2) mice
have been reported to be more sensitive to lipopolysaccharide
(LPS) induced lethal shock.12
Acute lung injury is characterised by neutrophilic inflam-
mation, increased expression of proinflammatory cytokines,
loss of epithelial and endothelial integrity, and the develop-
ment of interstitial oedema.13–16Intratracheal instillation of
LPS produces a well recognised model of acute lung injury
leading to the activation of alveolar macroghages, tissue infil-
tration of neutrophils, and interstitial oedema.17Although
inhalation of LPS has been reported to induce MT gene and
protein in the lung in vivo,18 19there is no evidence for the
direct contribution of MT in acute lung injury elicited by LPS.
etallothionein (MT) is a highly conserved, low
cysteine residues of MT bind and store metal ions, it
The development of acute lung injury requires several pul-
monary cell populations, transcriptional regulatory factors,
and proinflammatory molecules. Nuclear factor-kB (NF-kB)
activation and the subsequent expression of proinflam-
matory mediators also have an important role.20 21However,
the effects of MT on NF-kB activation is uncertain.22 23
This study was undertaken to determine whether intrinsic
MT has a role in protecting against acute lung injury induced
by LPS using MT-I/II null (MT (2/2)) mice and control wild
type (WT) mice. The mechanisms by which MT protects
against acute lung injury were also studied and its role in the
NF-kB pathway in vivo determined.
Animals and study protocol
MT (2/2) mice whose MT-I and MT-II genes had null
mutation and WT mice were provided by Dr Choo (Murdoch
Institute for Research into Birth Defects, Royal Children’s
Hospital, Australia).24They were of a mixed genetic back-
ground of 129 Ola and C57BL/6 strains. F1 hybrid mice were
mated with C57BL/6 mice and their offspring were back-
crossed to C57BL/6 for six generations in the National Insti-
tute for Environmental Studies (NIES; Tsukuba, Japan),
reproducing normally and displaying no overt abnormality in
physical state and behaviour. These mice were routinely bled
in the vivarium of NIES as previously described.10
MT (2/2) and WT mice were treated with vehicle or LPS
(E coli B55:05, Difco Lab, Detroit, MI, USA). For both
Abbreviations: IL, interleukin; KC, keratinocyte chemoattractant; LPS,
lipopolysaccharide; MCP-1, macrophage chemoattractant protein; MIP-
1a, macrophage inflammatory protein-1a; MT, metallothionein; NF-kB,
A Shimada, T Morita, M Sawada, K Nakamura, Department of
Veterinary Pathology, Faculty of Agriculture, Tottori University, Tottori,
C Sanbongi, Meiji Seika Kaisha Co, Saitama, Japan
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1062Takano, Inoue, Yanagisawa, et al