Regulation of macrophage inflammatory protein-1alpha mRNA by oxidative stress.

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-1α (MIP-1α), 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-1α 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-1α mRNA in macrophages and to determine the mechanism leading to increased expression. A rat alveolar macrophage cell line (NR8383) was exposed to HO 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-1α were markedly increased (3-fold) in these cells after 1 h of exposure to 0.5 mM HO, 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-1α mRNA with a maximal induction after 4 h of exposure (5-fold). Both HO and MQ-induced up-regulation of MIP-1α mRNA was suppressed by co-treatment with N-acetylcysteine, a synthetic antioxidant. Co-treatment with actinomycin D reduced the MQ induction of MIP-1α mRNA to a greater extent than the HO-induced increase. Transcription of the MIP-1α gene was increased by exposure to both HO and MQ. HO treatment also induced a marked increase of the MIP-1α mRNA half-life, indicating post-transcriptional stabilization. These observations indicate that an oxidative stress can regulate MIP-1α mRNA expression by two distinct mechanisms: transcriptional activation of the MIP-1α gene and post-transcriptional stabilization of MIP-1α mRNA.

Reactive oxygen species (ROS) 1 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(DeForge et al., , 1993Driscoll 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(Huang et al., , 1992bFarone 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 H 2 O 2 and MQ, and chemokine mRNA expression was measured in response to these oxidative stresses. In contrast to H 2 O 2 -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 2 O 2 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 2 O 2 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 2 O 2 induces MIP-1␣ mRNA expression by induction of MIP-1␣ gene transcription as well as by increasing the stability of the mRNA transcript.

EXPERIMENTAL PROCEDURES
Cells and Culture Conditions-Tissue culture supplies and related materials were purchased from Sigma unless otherwise stated. The rat alveolar macrophage (RAM) cell line, NR8383, was generously provided by Dr. R. Helmke (Helmke et al., 1987). Cells were cultured in RPMI 1640 medium supplemented with 5% equine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. Cells were grown in a humidified incubator at 37°C with 5% CO 2 .
Cell Treatment-Tissue culture plates were precoated with polyhydroxymethylacrylate to prevent cell adherence (Folkman and Moscona, 1987). Cells were plated at a density of 1 ϫ 10 6 cells/ml in serum-free RPMI for 4 h prior to treatment. MQ was dissolved in dimethyl sulfoxide and subsequently diluted in RPMI medium. A 30% H 2 O 2 stock was diluted in RPMI immediately before treatment. When used, MQ was added to tissue culture plates at a final concentration of 25 or 50 M for 1-6 h. Control cells were treated with the same concentrations of Me 2 SO as the MQ treatment group. Me 2 SO levels did not exceed 0.01%. No significant cytotoxicity was identified for any oxidant treatment used in this study as measured by trypan blue assays. When used, 1 mM N-acetylcysteine (NAC) was added to cells and preincubated for 1 h in serum-free RPMI before addition of MQ or H 2 O 2 . In other studies, RAM cells were treated with MQ or H 2 O 2 along with 5 g/ml actinomycin D or 5 g/ml cycloheximide for 1 or 4 h to inhibit cellular RNA and protein synthesis, respectively.
mRNA Half-life Determination-Following treatment of RAM cells with 25 M MQ for 4 h or 0.5 mM H 2 O 2 for 1 h as above, actinomycin D was added to the media to a final concentration of 5 g/ml. Cells were sampled at times indicated through 6 h, and the levels of MIP-1␣ mRNA were quantified by Northern analysis. The integrated band values, as determined by densitometry, were normalized to ␤-actin RNA.

RESULTS
Induction of MIP-1␣ mRNA by Oxidative Stress-RAM cells were exposed to H 2 O 2 up to 6 h, and steady-state mRNA levels encoding MIP-1␣ were measured by Northern analysis. Levels of MIP-1␣ mRNA were rapidly induced as early as 1 h following exposure to 0.5 mM H 2 O 2 , remained higher than controls after 4 h of exposure, and returned to the control level by 6 h (Fig. 1). H 2 O 2 at 0.1 mM did not induce a change in MIP-1␣ mRNA levels.
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 2 O 2 and O 2 continuously. MQ at both 25 and 50 M 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 M MQ and were markedly reduced at 50 M MQ (Fig. 2).
N-Acetylcysteine Suppresses the Increase of MIP-1␣ mRNA by Oxidative Stress-NAC is a synthetic antioxidant that can replenish intracellular glutathione levels (Meister and Anderson, 1983). RAM cells were pretreated with 1 mM NAC for 1 h and then challenged with either MQ or H 2 O 2 . NAC at this level completely eliminated the induction of MIP-1␣ mRNA induction by 25 and 50 M MQ (Fig. 3). In a similar fashion, NAC treatment partially attenuated the induction of MIP-1␣ mRNA by 0.5 mM H 2 O 2 at both 1 and 4 h postexposure (Fig. 4).
Effect of Cycloheximide and Actinomycin D on the Elevation of MIP-1␣ mRNA by Oxidative Stress-Steady-state levels of mRNAs may be modulated by transcriptional or post-transcriptional mechanisms. To determine the route through which MQ and H 2 O 2 elevate MIP-1␣ mRNA concentrations, we applied commonly used translational and transcriptional inhibitor assays as described previously (Shi et al., 1994a(Shi et al., , 1994b. RAM cells were exposed to 25 M MQ in the presence of 5 g/ml cycloheximide for 4 h to inhibit protein synthesis. Total cellular RNA was extracted and Northern analysis was performed. Cycloheximide alone caused a significant increase in MIP-1␣ mRNA levels, and did not appear to inhibit the induction of MIP-1␣ mRNA levels in response to MQ or H 2 O 2 (Fig. 5). These results only suggest that an increase of MIP-1␣ mRNA by MQ-induced oxidative stress may not be dependent on de novo protein synthesis.
Experiments were also performed in the presence of actinomycin D to inhibit transcription. RAM cells were exposed to 25 M MQ for 4 h or 0.5 mM H 2 O 2 for 1 h in the presence or the absence of 5 g/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 (ϳ60%) suppressed the increase of MIP-1␣ mRNA expression by H 2 O 2 (Fig. 6, B and D), suggesting that mRNA stability may also play an important role in the up-regulation of MIP-1␣ transcript levels.
Transcriptional Regulation of the MIP-1␣ Gene by an Oxidative Stress-Because transcriptional regulation of the MIP-1␣ gene by ROS was suggested by results obtained with actino- mycin D, we performed nuclear run-on transcriptional rate assays to confirm the role of this mechanism in regulation of mRNA expression. RAM cells were treated with 0 or 0.5 mM H 2 O 2 or 0 or 50 M MQ for 1 and 4 h. Cell nuclei were extracted, and transcription rates for the MIP-1␣ gene were determined as described under "Experimental Procedures." At 1 h posttreatment, transcription of the MIP-1␣ gene markedly increased following treatment with both 0.5 mM H 2 O 2 and 50 M MQ (Fig. 7). After 4 h, the transcriptional rate of the MIP-1␣ gene in MQ-treated cells was further increased; however, MIP-1␣ transcription in H 2 O 2 -treated cells had returned to near control levels. Hybridization to nonspecific, prokaryotic sequences (pBR322) was not evident. These results indicate that the observed induction of MIP-1␣ mRNA by both H 2 O 2 or MQ is at least in part caused by transcriptional activation of the gene.
Post-transcriptional Regulation of MIP-1␣ mRNA by H 2 O 2 -The MIP-1␣ mRNA 3Ј-untranslated region contains six copies of the reiterated AUUUA motifs (Shi et al., 1995) that are typically conserved in these regions of cytokines and growth factors mRNAs and are implicated in mRNA stability and translational control (Shaw and Kamen, 1986;Brewer, 1991). Our previous work suggested that the rapid up-regulation of FIG. 5. Effect of cycloheximide on MIP-1␣ mRNA levels in response to MQ and H 2 O 2 in rat alveolar macrophages. Left panels, cells were co-incubated with 0 or 25 M MQ and 0 or 5 g/ml cycloheximide for 4 h. Right panels, cells were treated with 0 or 0.5 mM H 2 O 2 and 0 or 5 g/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.

FIG. 6. Effect of actinomycin D (AD) on MIP-1␣ mRNA expression in response to MQ (A) and H 2 O 2 (B) in rat alveolar macrophages.
Cells were treated with 0 or 25 M MQ and 5 g/ml actinomycin D for 4 h (A) or with 0 or 0.5 mM H 2 O 2 and 5 g/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 2 O 2 , 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. MIP-1␣ mRNA by lipopolysaccharide treatment is the result of post-transcriptional regulation (Shi et al., 1995). The contribution of changes in MIP-1␣ mRNA stability to its increased expression in response to oxidative stress was evaluated by measuring MIP-1␣ mRNA half-life (t1 ⁄2 ). In the presence of actinomycin D, MIP-1␣ mRNA from untreated macrophages decayed quickly with a t1 ⁄2 of approximately 1 h (Fig. 8). H 2 O 2 treatment significantly increased the half-life of MIP-1␣ mRNA, with a t1 ⁄2 greater than 6 h. MQ treatment did not change the t1 ⁄2 of MIP-1␣ mRNA in comparison with the control. These observations are in agreement with the results of the actinomycin D (Fig. 6, A and B) and nuclear run-on studies (Fig. 7), suggesting that the induction of MIP-1␣ mRNA by MQ results from transcriptional activation, whereas the elevation of MIP-1␣ mRNA by H 2 O 2 is the result of both transcriptional activation of the MIP-1␣ gene and post-transcriptional regulation of the mRNA.

DISCUSSION
Induction of MIP-1␣ mRNA in alveolar macrophages by H 2 O 2 or MQ strongly suggests a role for ROS in the regulation of chemokine gene expression. Although H 2 O 2 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 2 O 2 -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, 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 reductionoxidation (redox) state of nuclear proteins. A number of sitespecific 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 5Ј-flanking region of Escherichia coli and Salmonella typhimurium catalase and alkylhydroperoxide reductase genes are recognized by a transactivator 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 5Ј-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 2 O 2 (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 FIG. 7. Transcriptional rate of the MIP-1␣ gene in rat alveolar macrophages exposed to H 2 O 2 or MQ. Cells were exposed to 0 or 0.5 mM H 2 O 2 or 50 M 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 2 O 2 -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. Actinomycin D was then added to a final concentration of 5 g/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 t1 ⁄2 from control or MQ-treated cells was approximately 1 h, whereas H 2 O 2 treatment increased the t1 ⁄2 to greater than 6 h. The results are representative of two independent experiments. 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 2 O 2 , 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 2 O 2 (Fig. 7). Although the 5Ј-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 2 O 2 . MQ and H 2 O 2 may also induce transcription of MIP-1␣ through cisacting element(s) like antioxidant response element. Whether the transcriptional induction of MIP-1␣ by MQ and H 2 O 2 is through an antioxidant response element-like element, an NF-B binding region, or other 5Ј 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 2 O 2 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 3Ј-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 t1 ⁄2 of less than 1 h, which is similar to that of control cells. On the contrary, MIP-1␣ mRNA from H 2 O 2 -treated cells has a t1 ⁄2 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 2 O 2 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 2 O 2 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 H 2 O 2and 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 2 O 2 , 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.