(Received for publication, November 15, 1995)
From the
Accumulation of inflammatory cells within the lung has been
implicated in oxidative injury. Recruitment of these cells to a tissue
site is a complex process that depends in part upon the local
expression of appropriate proinflammatory chemokines. Macrophage
inflammatory protein-1 (MIP-1
), a member of the CC subfamily
of chemokines, has been shown to contribute to monocyte/macrophage and
neutrophil chemotaxis and activation. Our previous work demonstrated
that MIP-1
mRNA expression in macrophages is induced by bacterial
endotoxin. The objective of this study was to test the hypothesis that
an oxidative stress alone may trigger expression of MIP-1
mRNA in
macrophages and to determine the mechanism leading to increased
expression. A rat alveolar macrophage cell line (NR8383) was exposed to
H
O
or menadione (2-methyl-1,4-naphthoquinone
(MQ)), a quinone compound that undergoes redox cycling and generates
reactive oxygen species continuously. Steady-state mRNA levels encoding
MIP-1
were markedly increased (3-fold) in these cells after 1 h of
exposure to 0.5 mM H
O
, remained higher
than control levels after 4 h, and decreased after 6 h. Similarly, MQ
(25 or 50 µM) caused a significant increase of MIP-1
mRNA with a maximal induction after 4 h of exposure (5-fold). Both
H
O
and MQ-induced up-regulation of MIP-1
mRNA was suppressed by co-treatment with N-acetylcysteine, a
synthetic antioxidant. Co-treatment with actinomycin D reduced the MQ
induction of MIP-1
mRNA to a greater extent than the
H
O
-induced increase. Transcription of the
MIP-1
gene was increased by exposure to both H
O
and MQ. H
O
treatment also induced a
marked increase of the MIP-1
mRNA half-life, indicating
post-transcriptional stabilization. These observations indicate that an
oxidative stress can regulate MIP-1
mRNA expression by two
distinct mechanisms: transcriptional activation of the MIP-1
gene
and post-transcriptional stabilization of MIP-1
mRNA.
Reactive oxygen species (ROS) ()have been implicated
in the pathogenesis of several lung diseases including adult
respiratory distress syndrome, asthma, chronic bronchitis, and lung
fibrosis (reviewed by Halliwell and Gutterage(1989)). Increased
production of ROS is believed responsible for tissue damage during
pulmonary inflammation (Southern and Powis, 1988). Inflammatory cells
such as neutrophils and macrophages play an important role in host
defense but may also contribute to tissue injury through the release of
tissuedamaging oxidants upon activation (reviewed by Sibille and
Reynolds(1990)). In addition, activated inflammatory cells also produce
mediators such as prostanoids and leukotrienes that can induce
bronchoconstriction. Thus, leukocytes that are recruited into the lung
and subsequently activated have profound effects on cells within the
lung and may propagate inflammation.
Recruitment of inflammatory
cells to a tissue site is a complex process that depends in part upon
the local expression of appropriate chemoattractant proteins termed
``chemokines'' (reviewed by Oppenheim et al.(1991)).
The chemokine superfamily can be subdivided into two subsets, which
differ with respect to the relative positioning of the first two
cysteines (CXC versus CC) at the N terminus.
Macrophage inflammatory protein-1 (MIP-1
), a member of the CC
family of chemokines, contributes to monocyte/macrophage and neutrophil
chemotaxis and activation. MIP-1
has been reported to be
chemotactic for mononuclear phagocytes, neutrophils, eosinophils,
basophils, and lymphocytes (Davatelis et al., 1988; Wolpe and
Cerami, 1989; Taub et al., 1993; VanOtteren et al.,
1994). MIP-1
also induces a respiratory burst in neutrophils and
plays a role in the control of hemopoietic stem cell proliferation
(Graham et al., 1990; Dunlop et al., 1992).
MIP-1
mRNA has been reported to be constitutively expressed at low
levels and up-regulated by inflammatory stimuli in pulmonary alveolar
macrophages (Shi et al., 1995). Recombinant MIP-1
administered to the lungs of rats elicits a localized neutrophilic
inflammatory response that can be neutralized with anti-MIP-1
antibody in vitro (Shanley et al., 1995). These
features make this chemokine a very likely participant in initiation of
inflammation by noxious stimuli.
A potential association
specifically between oxidative stress and chemokine expression has been
suggested (DeForge et al., 1992, 1993; Driscoll et
al., 1993). Because of their role in pulmonary defense and their
capacity for ROS production, alveolar macrophages are likely to be an
important target for oxidative stress. Previous studies have shown that
these cells are capable of expressing chemokines upon exposure to acute
or chronic inflammatory stimuli (Huang et al., 1992a, 1992b;
Farone et al., 1995; Shi et al., 1995). In this study
we utilized a rat alveolar macrophage cell line to test the hypothesis
that an oxidative stress alone can trigger expression of the chemokine
MIP-1 and thus contribute to early inflammation.
Macrophages
were exposed to both HO
and MQ, and chemokine
mRNA expression was measured in response to these oxidative stresses.
In contrast to H
O
-induced oxidative stress, two
major mechanisms are involved in quinone-induced cytotoxicity (reviewed
by Brunmark and Cadenas(1989)). First, quinone is reduced to the
hydroquinone or semiquinone radical by cellular reductase. The
semiquinone radical undergoes rapid autoxidation with the generation of
the parent quinone and concomitant formation of superoxide. The
hydroquinone reacts rapidly with superoxide to form
H
O
and the semiquinone (Fridovich 1981; Shi et al., 1994a). Secondly, most quinones, including MQ, also
react with cellular nucleophiles, such as thiols and amines, and cause
cellular damage (Chang et al., 1992; Shi et al.,
1993). We now demonstrate that both H
O
and MQ
induce MIP-1
mRNA expression in macrophages but through two
distinct mechanisms. The up-regulation of MIP-1
mRNA by MQ is
mediated through transcriptional activation of the MIP-1
gene.
H
O
induces MIP-1
mRNA expression by
induction of MIP-1
gene transcription as well as by increasing the
stability of the mRNA transcript.
Figure 1:
Induction of MIP-1
mRNA levels by H
O
in a rat alveolar macrophage
cell line, NR8383. Cells were incubated with 0, 0.1, or 0.5 mM H
O
in serum-free RPMI medium for 1, 4, or
6 h. Northern analysis was performed as described under
``Experimental Procedures'' using a MIP-1
cDNA as probe. Upper panels, autoradiogram of Northern blot hybridized with a
radiolabeled MIP-1
cDNA. Lower panels, the same membrane
hybridized with a mouse
-actin cDNA to indicate relative amounts
of hybridizable RNA per lane. The results are representative of two
independent experiments.
RAM cells were also
treated with a second source of reactive oxygen radicals, MQ, and
MIP-1 mRNA levels were again quantified. MQ undergoes redox
cycling and generates reactive oxygen species such as
H
O
and O
continuously. MQ at both
25 and 50 µM also caused an induction of MIP-1
mRNA
levels at 1 h, which persisted through 4 h of exposure with a maximum
5-fold increase over control levels (Fig. 2). After 6 h,
MIP-1
mRNA levels had fallen back to near control levels at 25
µM MQ and were markedly reduced at 50 µM MQ (Fig. 2).
Figure 2:
Induction of MIP-1 mRNA levels by MQ
in rat alveolar macrophages. Cells were incubated with 0, 25, or 50
µM MQ in serum-free RPMI medium for 1, 4, or 6 h. Northern
analysis was performed as described for Fig. 1. Upper
panels, autoradiogram of Northern blot hybridized with
radiolabeled MIP-1
cDNA. Lower panels, the same membrane
hybridized with a mouse
-actin cDNA to indicate relative amounts
of hybridizable RNA per lane. The results are representative of two
independent experiments.
Figure 3:
Effect of NAC on MIP-1 mRNA levels in
response to MQ in rat alveolar macrophages. Cells were incubated with 1
mM NAC for 1 h, followed by the addition of 0, 25, or 50
µM MQ and incubation for 4 h. Total cellular RNA was
extracted, and Northern analysis was performed as described for Fig. 1. A, upper panel, autoradiogram of
Northern blot hybridized with radiolabeled MIP-1
cDNA. Lower
panel, the same membrane hybridized with a mouse
-actin cDNA
to indicate relative amounts of hybridizable RNA per lane. B,
densitometric quantification of MIP-1
mRNA normalized to
-actin RNA. The results are representative of two independent
experiments.
Figure 4:
Effect of NAC on MIP-1 mRNA levels in
response to H
O
in rat alveolar macrophages. A, cells were incubated with 1 mM NAC for 1 h
followed by the addition of 0.5 mM H
O
for 1 or 4 h. Total cellular RNA was extracted and Northern
analysis was performed as described for Fig. 1. Upper
panels, autoradiogram of Northern blot hybridized with a
radiolabeled MIP-1
cDNA. Lower panels, the same membrane
hybridized with a mouse
-actin cDNA to indicate relative amounts
of hybridizable RNA per lane. B, densitometric quantification
of MIP-1
mRNA normalized to
-actin RNA. The results are
representative of two independent
experiments.
Figure 5:
Effect of cycloheximide on MIP-1 mRNA
levels in response to MQ and H
O
in rat alveolar
macrophages. Left panels, cells were co-incubated with 0 or 25
µM MQ and 0 or 5 µg/ml cycloheximide for 4 h. Right panels, cells were treated with 0 or 0.5 mM H
O
and 0 or 5 µg/ml cycloheximide for
4 h. Northern analysis was performed as described for Fig. 1. Upper panels, autoradiogram of Northern blot hybridized with
radiolabeled-MIP-1
cDNA. Lower panels, the same membrane
hybridized with a mouse
-actin cDNA to indicate relative amounts
of hybridizable RNA per lane. The results are representative of two
independent experiments.
Experiments
were also performed in the presence of actinomycin D to inhibit
transcription. RAM cells were exposed to 25 µM MQ for 4 h
or 0.5 mM HO
for 1 h in the presence
or the absence of 5 µg/ml actinomycin D. The elevation of
MIP-1
mRNA by MQ was almost completely blocked by co-incubation
with actinomycin D, suggesting that the increase by oxidative stress is
transcriptionally regulated (Fig. 6, A and C).
In contrast, actinomycin D only partially (
60%) suppressed the
increase of MIP-1
mRNA expression by H
O
(Fig. 6, B and D), suggesting that mRNA
stability may also play an important role in the up-regulation of
MIP-1
transcript levels.
Figure 6:
Effect of actinomycin D (AD) on
MIP-1 mRNA expression in response to MQ (A) and
H
O
(B) in rat alveolar macrophages.
Cells were treated with 0 or 25 µM MQ and 5 µg/ml
actinomycin D for 4 h (A) or with 0 or 0.5 mM H
O
and 5 µg/ml actinomycin D for 1 h (B). Total cellular RNA was extracted, and Northern analysis
was performed as described for Fig. 1. Upper panels,
autoradiogram of Northern blot hybridized with radiolabeled MIP-1
cDNA. Lower panels, the same membrane hybridized with a mouse
-actin cDNA to indicate relative amounts of hybridizable RNA per
lane. C and D, densitometric quantification of
MIP-1
mRNA normalized to
-actin RNA in response to MQ and
H
O
, respectively. Intensity of control
autoradiographic bands were defined as 1 in order to compare fold
changes in MIP-1
mRNA in the absence or the presence of
actinomycin D. The results are representative of two independent
experiments.
Figure 7:
Transcriptional rate of the MIP-1
gene in rat alveolar macrophages exposed to H
O
or MQ. Cells were exposed to 0 or 0.5 mM H
O
or 50 µM MQ for 1 and 4 h.
Nuclei were then extracted, and nuclear run-on assays were performed as
described under ``Experimental Procedures.'' Equivalent
amounts of radiolabeled RNA prepared from nuclei isolated from
untreated or H
O
- or MQ-treated cells were
hybridized with denatured MIP-1
, glyceraldehyde-3-phosphate
dehydrogenase (control) cDNA, or pBR322 plasmid DNA. The results are
representative of two independent
experiments.
Figure 8:
Influence of HO
and MQ on the half-life (t
) of MIP-1
mRNA in rat alveolar macrophages. Cells were left untreated (Control) or treated with 0.5 mM H
O
for 1 h or 25 µM MQ for 4 h. Actinomycin D was then
added to a final concentration of 5 µg/ml, and at the times
indicated, total RNA was isolated and Northern analysis was performed
as described in Fig. 1A, upper panels,
autoradiogram of Northern blot hybridized with radiolabeled-MIP-1
cDNA. Lower panels, the same membrane hybridized with a mouse
-actin cDNA to indicate relative amounts of hybridizable RNA per
lane. B, densitometric quantification of the decay of
MIP-1
mRNA normalized to
-actin RNA. The MIP-1
mRNA t
from control or MQ-treated cells was
approximately 1 h, whereas H
O
treatment
increased the t
to greater than 6 h. The results
are representative of two independent
experiments.
Induction of MIP-1 mRNA in alveolar macrophages by
H
O
or MQ strongly suggests a role for ROS in
the regulation of chemokine gene expression. Although
H
O
is a direct source of oxidative stress, MQ
undergoes redox cycling, leading to a gradual increase in ROS levels
(Chang et al., 1992; Shi et al., 1993). Both MQ- and
H
O
-induced increases in MIP-1
mRNA were
suppressed by the antioxidant NAC, further confirming that oxidative
stress alone could influence MIP-1
expression. Although a number
of other recent studies have explored the possible modulation of
chemokine expression by ROS (DeForge et al., 1992, 1993), most
used an endotoxin challenge as a stimulus for oxidant stress. The
significance of the present study is that it directly links the ROS to
increased MIP-1
mRNA expression in the absence of endotoxin.
Reactive oxygen species are now known to activate gene expression
through modulation of the intracellular reduction-oxidation (redox)
state of nuclear proteins. A number of site-specific DNA binding
proteins have been identified whose redox state affects binding
ability. Oxidative activation of a transcription regulatory protein was
first identified in prokaryotes. cis-Acting elements in the
5`-flanking region of Escherichia coli and Salmonella
typhimurium catalase and alkylhydroperoxide reductase genes are
recognized by a trans-activator termed OxyR (Christman et
al., 1989). Both reduced and oxidized OxyR binds the catalase
promoter, but only the oxidized form activates transcription (Storz et al., 1990). In mammalian cells, ROS have also been
implicated in the activation of the transcription factors c-Fos and
c-Jun (Abate et al., 1990), NF-B/Rel (Schreck et
al., 1991). A cis-acting regulatory sequence, the
antioxidant response element, in the 5`-flanking region of the rat
glutathione transferase Ya subunit gene and rat NAD(P)H:quinone
reductase, has also been identified to be responsive to a variety of
redox cycling xenobiotics and H
O
(Rushmore et al., 1991; Favreau and Pickett, 1992). Constitutive
antioxidant response element-binding factor(s) have been identified
that are activated by ROS (Favreau and Picket, 1992). It has been
reported that a neonatal rat lung protein forms specific complexes with
catalase mRNA in a redox-sensitive manner. Neonatal rats exposed to
hyperoxia show increased lung catalase mRNA stability associated with a
larger proportion of catalase mRNA binding protein in oxidized form
than from lungs of air-breathing neonatal rats (Clerch et al.,
1991; Clerch and Massaro, 1992). These reports indicate that the
intracellular redox state is crucial in the control of either active
transcription factors or redox-sensitive proteins. Oxidant-induced
conformational changes of regulatory proteins may influence a spectrum
of genes by initiating transcription and/or stabilizing specific mRNAs.
We performed inhibitor studies to identify whether increases in
MIP-1 mRNA levels by oxidative stress were dependent on new RNA
synthesis or de novo protein synthesis. Cycloheximide alone
was able to markedly stimulate the accumulation of MIP-1
mRNA (Fig. 5), possibly suggesting that MIP-1
gene may be
controlled either directly or indirectly by a repressor protein with a
short half-life. Co-incubation with actinomycin D almost completely
eliminated the increase of MIP-1
mRNA by MQ (Fig. 6A) and partially by H
O
,
suggesting that the transcriptional regulation of the MIP-1
gene
is involved. This suggestion was confirmed with nuclear run-on assays,
which demonstrated increased transcriptional rates induced by MQ and
H
O
(Fig. 7). Although the 5`-flanking
region of rat MIP-1
is presently unavailable, its murine
counterpart contains an NF-
B-like binding region (Grove and Plumb,
1993). NF-
B is a multi-subunit transcription factor that can
rapidly activate the expression of genes involved in inflammation and
the acute phase immune response (Baeuerle and Baltimore, 1988). As
described above, NF-
B has been previously reported to be activated
by oxidative stress, including H
O
. MQ and
H
O
may also induce transcription of MIP-1
through cis-acting element(s) like antioxidant response
element. Whether the transcriptional induction of MIP-1
by MQ and
H
O
is through an antioxidant response
element-like element, an NF-
B binding region, or other 5`
sequences deserves further study.
Stability of mRNA transcripts also
plays an important role in the regulation of proinflammatory genes.
Changes in turnover rate can affect steady-state levels over a
relatively short period of time. As an example, the rapid,
hyperoxia-induced elevation of lung catalase mRNA in neonatal rats is
due to enhanced stability of its mRNA but not an increased rate of
transcription (Clerch et al., 1991). In the present study,
MIP-1 mRNA was elevated by H
O
and MQ in an
exceedingly short period of time (1 h), suggesting that MIP-1
mRNA
turnover might be influenced by oxidative stress. Also of note, the
3`-untranslated regions of the rat MIP-1
mRNA contains six copies
of reiterated AU-rich motifs (Shi et al., 1995), implicated in
mRNA stability and translational control (Shaw and Kamen, 1986; Brewer,
1991). A cytoplasmic protein termed adenosine-uridine binding factor
has been shown to bind specifically to the AUUUA motifs of in vitro transcribed RNAs and form exceptionally stable complexes (Malter,
1989). Moreover, the binding of adenosine-uridine binding factor to RNA
templates is also redox-sensitive (Malter and Hong, 1991). To
distinguish between an increase in transcriptional rate and
stabilization of pre-existing transcripts of MIP-1
, we performed
an RNA decay assay. MQ-induced elevation of MIP-1
mRNA has a t
of less than 1 h, which is similar to that of
control cells. On the contrary, MIP-1
mRNA from
H
O
-treated cells has a t
greater than 6 h (Fig. 8). Thus, the induction of
MIP-1
mRNA by MQ is primarily through transcriptional activation
of the MIP-1
gene, whereas the effect of H
O
is through both transcriptional activation of the MIP-1
gene
and the stabilization of MIP-1
mRNA. Post-transcriptional
regulation of MIP-1
mRNA in macrophages has been previously
reported following stimulation by bacterial endotoxin (Shi et
al., 1995), which can also generate ROS (Adamson and Billings,
1990; Yoshikawa, 1990). It is noteworthy that two distinct mechanisms
are involved in the regulation of MIP-1
gene expression; the one
chosen is dependent on the specific stimulus. Enhanced stability of
MIP-1
mRNA by H
O
may be due to an mRNA
binding protein whose confirmation, synthesis, or degradation is
changed during an oxidative stress.
In conclusion, the present study
demonstrates that an HO
- and MQ-generated
oxidative stress rapidly elevates MIP-1
mRNA expression in
macrophages. The up-regulation of MIP-1
by MQ was apparently
mediated through transcriptional activation of the MIP-1
gene.
H
O
, on the other hand, induced MIP-1
mRNA
expression by two mechanisms: transcriptional activation of the
MIP-1
gene and stabilization of the MIP-1
mRNA.