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Originally published online as doi:10.1189/jlb.0303104 on August 1, 2003

Published online before print August 1, 2003
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(Journal of Leukocyte Biology. 2003;74:908-915.)
© 2003 by Society for Leukocyte Biology

Antibiotic cyclic AMP signaling by "primed" leukocytes confers anti-inflammatory cytoprotection

Kazuhiro Abeyama*,1, Ko-ichi Kawahara*, Satoshi Iino*, Takashi Hamada*, Shin-ichiro Arimura*, Kenji Matsushita{dagger}, Toshihiro Nakajima{ddagger} and Ikuro Maruyama*,1

Departments of
* Laboratory and Molecular Medicine, Faculty of Medicine, and
{dagger} Operative Dentistry and Endodontology, Dental School, Kagoshima University, Japan; and
{ddagger} Department of Genome Science, Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki, Japan

1Correspondence: Department of Laboratory and Molecular Medicine, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, Japan, 890-8520. E-mail: k-abeyam{at}m3.kufm.kagoshima-u.ac.jp


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ABSTRACT
 
The mechanism underlying anti-inflammatory effects of macrolide antibiotics remains uncertain. In this study, we first show the evidences concerning the possible link between leukocytic cyclic adenosine monophosphate (cAMP) signaling and the mechanism of anti-inflammatory, cytoprotective actions of macrolides. The clinical range of macrolides (i.e., erythromycin, roxithromycin, and clarithromycin) preferentially inhibited nuclear factor-{kappa}B activation mediated by reactive oxygen intermediates, inducing cAMP-dependent signaling [i.e., cAMP and cAMP-responsive element-binding protein (CREB)] by "primed" but not "resting" leukocytes. In this context, cAMP/CREB inhibition with adenosine 3':5'-cyclic monophosphothioate, rp-isomer (rp-cAMPs) and CREB decoy oligonucleotides reduced the anti-inflammatory actions of macrolides. These results thus indicate that macrolide-induced cAMP/CREB signaling, selectively by primed leukocytes, plays a major role in the mechanism of anti-inflammatory actions of macrolides.

Key Words: NF-{kappa}B • cytokines • transcriptional factors • signal transduction • reactive oxygen intermediates (ROI)


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INTRODUCTION
 
Erythromycin (EM), clarithromycin (CAM), and roxithromycin (RXM), which are 14-member lactone-ring macrolide antibiotics, have been shown to be effective in treating patients with chronic respiratory diseases {i.e., chronic sinusitis, bronchial asthma, chronic bronchitis, and diffuse panbronchiolitis (DPB); ref. [1 ]}. The mechanism of the actions is recently considered as anti-inflammatory effects characterized by the inhibition of nuclear factor (NF)-{kappa}B-dependent events [e.g., productions of proinflammatory tumor necrosis factor {alpha} (TNF-{alpha}), interleukin (IL)-1ß, IL-6, and IL-8] rather than the antimicrobial effects [1 2 3 4 5 ]. However, the further molecular mechanism, how macrolides inhibit NF-{kappa}B to induce anti-inflammatory actions, still remains unclear.

The transcriptional factor NF-{kappa}B has been activated in response to various inflammatory triggers including bacterial components [i.e., lipopolysaccharide (LPS), bacterial CpG DNAs], inflammatory cytokines (i.e., TNF-{alpha}, IL-1ß), DNA-damaging chemicals, and short-wave photon (i.e., UV, X-ray) [6 7 8 9 10 11 12 13 14 ]. With respect to the molecular mechanism, reactive oxygen intermediates (ROI) have played a central role in certain NF-{kappa}B-dependent, proinflammatory events, which could be inhibited by antioxidants [14 15 16 17 18 ]. In this study, we first show the evidence that a clinical range of macrolides (i.e., EM, CAM, and RXM) preferentially inhibited ROI-mediated NF-{kappa}B activation through the induction of cyclic adenosine monophosphate (cAMP)/cAMP response element-binding protein (CREB) signaling.


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MATERIALS AND METHODS
 
Reagents
LPS (Escherichia coli serotype O111: B4), EM, N-L-acetyl-cystein (NAC), dibutyryladenosine 3':5' cAMP (db-cAMP), N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H89), adenosine 3':5'-cyclic monophosphothioate, rp-isomer (rp-cAMPs), N{omega}-nitro-L-arginine methylester (L-NAME), thrombin, and mastoparan were purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant TNF-{alpha}, IL-1ß, and IL-10 were purchased from R&D Systems (Minneapolis, MN). CAM was purchased from Wako Chemical Co. (Tokyo, Japan). RXM was kindly provided from Eisai Pharmaceutical Co. (Tokyo, Japan). Dichlorofluorescein diacetate (DCFH-DA) was purchased from Molecular Probes (Eugene, OR).

Cells and cell culture
Human peripheral blood leukocytes (PBL), human leukemia line, THP-1, and human lung alveolar epithelial cell line A549 were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. To assess the leukocyte-induced tissue damage, A549 cells were grown to confluence on the 24-well culture plate in the presence of transwell chambers (12 mm diameter, 0.45 µm pore-sized Millicell-HA, Millipore, Bedford, MA), which contained indicated number of leukocytes (coculture system). Cell viability was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as described previously [19 ]. To prepare Tax-expressing THP-1 cells, cells were transfected with Tax-expression vector pH2R40M and pH2Rneo as a control (Riken, Tsukuba, Japan).

Enzyme-linked immunosorbent assay (ELISA) and fluorescent measurement of ROI
The levels of cytokines were assayed by using cytokine-specific ELISA kits for IL-6, IL-8 (Otsuka Assay Co., Tokushima, Japan), and IL-10 (R&D Systems). The levels of intracellular cAMP were also assayed by using ELISA kit for cAMP (R&D Systems). The formation of intracellular H2O2 as ROI production was determined using DCFH-DA as described previously [14 , 20 ]. Briefly, cells were preloaded with 5 µM DCFH-DA in culture medium for 30 min and then incubated for an additional 30 min under the various conditions as indicated. Fluorescent measurements were performed using fluorescein-activated cell sorter (FACS) and fluorescent photometer.

Characterization of cell death pattern
To characterize cell death pattern, DNA ladder assays were performed as described previously [21 ]. Briefly, 106 cells were lysed in 100 µl 10 mM Tris-HCl buffer (pH 7.4) containing 10 mM EDTA and 0.5% Triton X-100. After centrifugation for 5 min at 15,000 rpm, supernatant samples were treated with RNase A and proteinase K. Subsequently, 20 µl 5 M NaCl and 120 µl isopropanol were added to the samples and kept at -20ºC for 6 h. Following centrifugation for 15 min at 15,000 rpm, pellets were dissolved in 20 µl 10 mM Tris-HCl, 1 mM EDTA, as loading samples. To assay DNA fragmentation pattern, samples were loaded on 1.5% agarose electrophoresis.

Assays to assess the activities of NF-{kappa}B and CREB
Electrophoretic mobility shift assays (EMSAs) for selected transcriptional factors were performed as described previously [9 ]. Briefly, 5 µg nuclear proteins were incubated with 32P-radiolabeled, double-stranded oligodeoxynucleotides (ODNs) containing the sequences as follows: NF-{kappa}B, 5'-AGTTGAGGGGACTTTCCCAGGC-3', and CREB, 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3'; octamer (OCT-1), 5'-TGTCGAATGCAAATCACTAGAA-3'. Mixtures were then analyzed on native 5% polyacrylamide gels using Tris-bolic acid/EDTA buffer. The DNA-binding activities of OCT-1 nuclear protein, a member of the OCT family, which is apparently ubiquitous in mammalian cells, normalized nuclear extracts [22 ]. To measure NF-{kappa}B- and CREB-dependent transcription, reporter gene assays were performed as described previously [10 ]. Briefly, cells were transfected with the transcriptional factor-specific reporter constructs [i.e., 3x NF-{kappa}B heat-stable secretory alkaline phosphate (SEAP), and 3x cAMP-responsive element (CRE) SEAP]. After transfection and subsequent cell incubation under indicated conditions, the enzyme activities of SEAP were measured (Clontech Laboratory, Palo Alto, CA).

CRE decoy ODN treatment
A 24-mer single-stranded oligonucleotide containing a palindromic CRE sequence, which self-hybridizes to form duplex/hairpin, was synthesized as described previously [23 ]. The sequences of CRE decoy and scrambled control decoy are as follows: CRE decoy ODN, 5'-TGACGTCATGACGTCATGACGTCA-3'; scrambled control ODN, 5'-CTAGCTAGCTAGCTAGCTAGCTAG-3'.

DNA transfection
To deliver gene constructs and decoy ODNs, cationic liposome, Superfect (Qiagen, Valencia, CA), was used in the decoy ODN treatment.

Data processing
The values were averaged, and the SD of triplicate experiments was calculated. Statistical significance was analyzed by unpaired (two-tail) t-test. A level of P < 0.05 was considered as a significant difference.


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RESULTS
 
Macrolides preferentially inhibit ROI-mediated "proinflammatory events"
The first set of experiments showed cytokine profiles of LPS-treated PBL in the presence of indicated macrolides (Table 1 ). Macrolides (i.e., EM, RXM, and CAM) significantly inhibited LPS-induced, highly increased cytokine productions (i.e., IL-6 and IL-8) by PBL (Table 1) as well as by a leukemia line THP-1 (data not shown). By contrast, each macrolides tested in this study facilitated production of anti-inflammatory cytokine IL-10 by PBL but not by THP-1 (data not shown) in the presence of LPS (Table 1) . In addition to cytokine productions, the macrolides also inhibited LPS-induced, rapid ROI productions (Table 1) . We then examined whether macrolides could inhibit cytokine-inducible NF-{kappa}B-dependent transcription using THP-1. As shown in Figure 1 , LPS-induced, NF-{kappa}B-dependent transcriptional activities were almost completely inhibited by antioxidant NAC, suggesting that LPS-induced NF-{kappa}B activities required ROI as an initial step of "proinflammatory signaling". Similarly to antioxidant effects, macrolides had significant inhibitory effects on LPS-induced ROI and resultant NF-{kappa}B-dependent transcription (Fig. 1) . In EMSA assays for nuclear extracts of PBL, LPS-induced, rapid DNA-binding activities of NF-{kappa}B (shown within 1 h) were significantly inhibited by macrolides as well as by NAC (Fig. 2A , left; the effects of EM and CAM were not shown), whereas DNA-binding activities of OCT-1 protein used as internal controls for EMSA [22 ] were not affected. The inhibitory effect of macrolide (RXM) on the NF-{kappa}B binding was sustained up to 8 h (Fig. 2B) . With respect to the mechanism, LPS-induced ROI is induced by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation through the interaction between LPS and Toll-like receptor 4 [24 ], which plays a causative role for NF-{kappa}B activation [14 15 16 17 18 , 24 ]. Macrolides are thus considered to exert anti-inflammatory effects via intrinsic antioxidant properties, at the level of reduced generation of ROI or dampening the cellular response to ROI. In this context, H2O2-induced, rapid NF-{kappa}B activities were significantly inhibited by NAC but not by macrolide (Fig. 2A , right), suggesting that macrolides inhibited NADPH oxidase-mediated ROI production by "primed" leukocytes but not rapid, cellular responses to ROI, which differed from the common antioxidant effects. In contrast to LPS-induced events, human T cell lymphotropic virus type I (HTLV-I) Tax-induced NF-{kappa}B activation [21 ], showing no significant ROI production, was unaffected by macrolides (Fig. 3 ) as well as by NAC (data not shown). Consistent with these results, anti-inflammatory spectrum of macrolide (RXM) based on NF-{kappa}B-specific reporter gene analyses also revealed that the tested macrolides preferentially inhibited ROI-mediated NF-{kappa}B activation by proinflammatory, primed leukocytes (Table 2 ).


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  		Table 1. Impact of Macrolide Antibiotics on LPS-Induced Productions of Cytokine and ROI by PBL



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  		Figure 1. Effect of antioxidant and macrolides on LPS-induced, proinflammatory signalings by THP-1. (Left) LPS-induced, proinflammatory signalings (ROI and NF-{kappa}B-dependent transcription) can be quenched in the presence of antioxidant NAC. (Right) Dose-dependent effects of macrolides on LPS-induced, proinflammatory signalings. For ROI analysis, fluorescent measurement of cellular H2O2 production was performed as in Materials and Methods, following 1 h incubation of leukocytes under various stimuli as indicated (LPS, 1 µg/ml; NAC, 1 mM). Data shown are the mean ± SD of fluorescense intensities from triplicate samples. For the measurement of NF-{kappa}B-dependent, transcriptional activities, reporter gene assays were performed as described in Materials and Methods. Briefly, cells transfected with 3x NF-{kappa}B reporter gene were incubated under various conditions as indicated for 12 h. Data shown are the mean ± SD of fold SEAP enzymatic activities from triplicate samples.



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  		Figure 2. Inhibitory effects of macrolides on LPS-induced, DNA-binding activities of NF-{kappa}B. EMSA assays for nuclear extracts of PBL were performed as described in Materials and Methods. (A) The effects of RXM (indicated dose) and NAC (1 mM) on NF-{kappa}B activities induced by 1 µg/ml LPS (left panel) or by 0.1 mM H2O2 (right panel). Cells were treated as indicated for 1 h. Data shown are shifted bands for NF-{kappa}B and OCT-1. NS, Nonspecific bands. (B) Time-course analysis of LPS-induced NF-{kappa}B activities in the presence or absence of 10 µM RXM.



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  		Figure 3. No inhibitory effect of ROI-independent NF-{kappa}B activities by leukocytes carrying HTLV-I–Tax gene. (A) FACS analysis of HTLV–Tax expression. THP-1 transfected with HTLV-I–Tax gene shows significant expression of Tax protein compared with control transfectant (Cont). (B) No additional ROI production by Tax transfectant. Data shown are ROI productions of control transfectant in the presence or absence of 1 µM LPS for 1 h and of Tax transfectant. (C) The effect of macrolides on Tax-induced NF-{kappa}B activities. Tax tranfectants were incubated for 1 h in the presence or absence of 10 µM RXM. Note that HTLV-I–Tax-transfected THP-1 shows macrolide-insensitive character on NF-{kappa}B activation.


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  		Table 2. Anti-inflammatory Spectrum of Macrolides on Proinflammatory Stimuli

To further examine whether macrolides could protect tissues from "injuries" as the culmination of inflammatory events (i.e., ROI production and NF-{kappa}B activation), we originally established a quantitative assay for inflammatory tissue damage using the leukocyte coculture system. As shown in Figure 4A , "LPS-primed" leukocytes (THP-1 and PBL) significantly induced apoptosis of alveolar epithelial A549 cells, which could be prevented by the indicated range of macrolides (Fig. 4B) or by NAC but not by inhibitor for NOS, L-NAME (Fig. 4C) . These evidences suggested that the A549-killing effects by primed leukocytes, which were mediated by LPS-induced ROI rather than reactive nitrogen intermediates induced by NF-{kappa}B-dependent, inducible NOS could be prevented by macrolides. In the mechanism of cytoprotection, macrolides were less effective to prevent A549 cells from lethal injuries by chemical oxidants (i.e., hydrogen peroxide and tert-butylperoxide) or ionizing radiation (data not shown). Taken together, these macrolides are thus considered to exert anti-inflammatory and cytoprotective effects through inhibition of ROI-mediated events (i.e., NF-{kappa}B activation and cell injury) by primed leukocytes. The antioxidant-like effects of macrolides against primed leukocytes thus clinically contribute not only to inhibition of NF-{kappa}B-dependent cytokine production but also to protection from inflammation-induced tissue damage.



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  		Figure 4. Cytoprotective effects of macrolides on cellular injuries by primed leukocytes. (A) Establishment of quantitative assay for inflammatory tissue damage using the leukocyte coculture system. A549 cell monolayer in the presence of transwell chambers containing the indicated number of leukocytes was incubated for 16 h, and then A549 cell viability was quantified by using MTT assay (left). Data shown are representative of three independent experiments, showing percent viabilities (mean±SD) of nontreated A549 cells. Statistically significant differences (*, P<0.05; **, P<0.01) compared with A549 alone. (Right) Apoptotic characteristics of A549 cell death induced by primed leukocytes (DNA ladder assay). (B) Impact of macrolides on the A549-killing effect by primed leukocytes. Statistically significant differences (*, P<0.05; **, P<0.01) compared with THP-1 coculture group without added macrolides. Statistically significant differences (#, P<0.05; ##, P<0.01) compared with PBL coculture group without added macrolides. (C) Effects of various free radical scavengers on A549-killing effects of primed leukocytes. A549 cells were cocultured with indicated leukocytes in the presence of an antioxidant (1 mM NAC) or an inhibitor for nitric oxide synthase (NOS; 1 mM L-NAME). Statistically significant differences (*, P<0.05; **, P<0.01) compared with THP-1 coculture group in the absence of NAC and L-NAME. Statistically significant differences (#, P<0.05) compared with PBL coculture group without any treatments.

Involvement of cAMP/CREB signal activation in the processes of anti-inflammatory actions of macrolides
In the mechanism of antioxidant-like and anti-inflammatory effects of macrolides, two lines of evidences led us to postulate that macrolides might facilitate intracellular cAMP productions to inhibit proinflammatory signaling by primed leukocytes. First, macrolides could reduce the severity of airway hyper-responsiveness, similarly to the actions of cAMP-increasing agents [25 ]. Second, cAMP-increasing agents also had anti-inflammatory actions [26 27 28 29 30 31 32 ]. We thus examined whether macrolides could induce cAMP production by leukocytes. As shown in Figures 5 and 6C , macrolides, indeed, could rapidly increase intracellular cAMP levels and CREB activities by LPS-primed leukocytes (PBL and THP-1) but not by "quiescent" leukocytes (Fig. 4) . With respect to the mechanism, each macrolide tested in this study could additionally increase intracellular cAMP, even in the presence of forskolin (Fig. 4B) , prostaglandin E1, or an inhibitor for cAMP-degrading phosphodiesterase, 3-isobutyl-1-methylxanthine (data not shown). These results suggested possibly distinct mechanisms of cAMP-increasing effects between macrolides and other cAMP-increasing drugs such as pentoxyfillin, iloplast, and roliplam [28 29 30 31 ].



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  		Figure 5. Effects of macrolides on intracellular cAMP levels. (A) Leukocytes (THP-1 and PBL) were incubated with 1 µg/ml LPS in the presence of macrolides (10 µM each) for the indicated time course, and then, ELISA measured the amounts of intracellular cAMP, as described in Materials and Methods. (B) Enhanced cAMP production by primed leukocytes with LPS and forskolin but not by quiescent leukocytes in the presence of macrolides (representative data shown are the effect of RXM). THP-1 cells were incubated under various conditions as indicated (LPS, 1 µg/ml; forskolin, 25 µg/ml) for 120 min in the presence or absence of 10 µM RXM. Statistically significant differences (**, P<0.01) compared with LPS-treated group without added macrolide. Statistically significant differences (##, P<0.05) compared with forskolin-treated group in the absence of macrolide.



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  		Figure 6. Functional role of cAMP/CREB signaling in the mechanism of anti-inflammatory actions of macrolides. (A and B) The linkage between actions of macrolides and inhibitory effect of cAMP-dependent signaling on ROI production by primed leukocytes. (A) THP-1 cells were incubated under various conditions as indicated (LPS, 1 µg/ml; RXM, 10 µM; db-cAMP, 50 µM) in the presence or absence of 150 µM rp-cAMPs. Data shown are representative of three independent experiments, showing fluorescence intensities (mean±SD, n=3). Statistically significant differences (##, P<0.01) compared with LPS plus RXM group. Statistically significant differences (**, P<0.01) compared with LPS plus db-cAMP group. (B) The effect of modulation of cAMP-dependent signalings on A549-killing effect by primed leukocytes. A549 cells were cocultured with LPS-treated THP-1 cells at a density of 106 under indicated conditions in the presence or absence of rp-cAMPs. Cell viabilty of A549 cells was measured as shown in Figure 3 . Statistically significant differences (¶, P<0.05) compared with THP-1 coculture plus RXM group. Statistically significant differences (*, P<0.05) compared with THP- 1 coculture alone. Statistically significant differences (#, P<0.05) compared with THP-1 coculture plus db-cAMP group. (C) Effect of macrolide antibiotics on CREB activity. (Upper) CREB–DNA binding activity in peripheral blood mononuclear cells (EMSA). Cells were incubated as indicated for 1 h, and then, nuclear extracts were analyzed. (Lower) CREB-mediated transcriptional activity in THP-1 cells (reporter gene assay). Cells transfected 3x the CRE reporter gene were incubated as indicated for 12 h, and then, reporter gene assay was performed. Statistically significant differences (*, P<0.05) compared with LPS-treated group without added macrolides. (D) Effect of CREB inhibition on the macrolide actions. Monocytes were transfected with CREB decoy oligonucleotides or scrambled control and then ROI generations by THP-1 or PBL (top), and IL-10 productions by PBL (middle) in the presence of 1 µg/ml LPS and 10 µM RXM were measured as described in Materials and Methods. To examine the efficacy of decoy ODNs, THP-1 cells were cotransfected with CREB-specific reporter gene and decoy ODNs, and then reporter SEAP assay was performed as described in Materials and Methods (bottom). Statistically significant differences (*, P<0.05) compared with LPS plus RXM without added ODNs.

We then examined the possible linkage between the macrolide effects (i.e., antioxidant-like and anti-inflammatory effects) and cAMP/CREB signaling pathway. Likewise macrolides, a membrane-permeable agonistic analog of cAMP (db-cAMP) inhibited LPS-induced ROI (Fig. 6A) and also reduced A549-killing effects of primed leukocytes in coculture experiment (Fig. 6B) . Furthermore, the antioxidant-like and cytoprotective effects of macrolides were almost completely reversed in the presence of an inhibitor of cAMP-dependent signalings, rp-cAMPs (Fig. 6A and 6B) . These results thus suggested that macrolides induced cAMP-signaling pathway specifically by primed leukocytes, which essentially contributed to the antioxidant-like effects of macrolides.

However, Table 1 also demonstrated a partial inhibitory effect of macrolides on ROI-independent NF-{kappa}B transcriptional activities by primed leukocytes treated with TNF-{alpha} or IL-1ß (Table 1.) . In this context, the increased cAMP has been reported to inhibit NF-{kappa}B-dependent transcription without interfering NF-{kappa}B nuclear translocation under certain conditions [32 ]. Consistent with the evidences, macrolides failed to inhibit TNF-{alpha}-induced, rapid activation of NF-{kappa}B (i.e., nuclear translocation and DNA binding, data not shown), although macrolides increased cAMP levels (3.2±0.6 folds) and partially inhibited NF-{kappa}B-dependent transcription, implying additional inhibitory effects of cAMP on NF-{kappa}B activities. We thus examined the functional role of downstream, CREB-dependent transcription in the mechanism of anti-inflammatory actions of macrolides. For this aim, we used CRE decoy ODN containing CRE sequence to block CREB-mediated, transcriptional activities. In CREB-dependent reporter SEAP assay, CREB activities by macrolides were almost completely blocked by CRE decoy but not by scrambled decoy (Fig. 6D , bottom). However, the pretreatment with CRE decoy did not affect inhibition of ROI production (Fig. 6D , top) or cAMP production (data not shown) by macrolides. By contrast, the macrolide-induced IL-10 productions by PBL were significantly inhibited by CRE decoy ODN but not by scrambled decoy (Fig. 6D , middle). These results indicated that antioxidant-like effects of macrolides on primed leukocytes, which were almost totally cAMP-dependent, did not involve downstream CREB activation in the mechanism, whereas macrolide-induced, anti-inflammatory cytokine productions (i.e., IL-10) by primed leukocytes were cAMP/CREB-dependent. In the study using recombinant IL-10, macrolide-inducible IL-10 could also significantly inhibit even ROI-independent NF-{kappa}B activities (e.g., HTLV–Tax-induced NF-{kappa}B) as well as the dependent mechanism without affecting ROI productions by primed leukocytes (data not shown), suggesting CREB-dependent gene products (e.g., IL-10) might contribute to additional anti-inflammatory effects of macrolides, which thus have anti-inflammatory actions characterized by NF-{kappa}B inhibition at multisteps through cAMP-dependent ROI inhibition and CREB-mediated anti-inflammatory protein productions, specifically by primed leukocytes.


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DISCUSSION
 
Clinical relevance
The present study showed the mechanism of anti-inflammatory actions of 14-member lactone-ring macrolide antibiotics such as EM, RXM, and CAM. Furthermore, our preliminary, clinical studies revealed the effectiveness in the treatment of DPB patients with macrolides. For example, treatment with RXM, 300 mg orally daily for 2 weeks, ameliorated the clinical symptoms and significantly reduced the production of IL-6 (18.5±8.7 vs. 12.6±5.9 pg/ml, P<0.05, n=12) and IL-8 (75.1±65.5 vs. 28.1±22.1 pg/ml, P<0.05, n=12).

In the mechanism of anti-inflammation, the induced cAMP/CREB signaling by macrolides plays a causativeal role for the inhibition of proinflammatory events (ROI production and NF-{kappa}B activation), which differs from nonsteroidal anti-inflammatory drug (NSAID) actions. In our observation, several NSAIDs could not induce cAMP/CREB signaling or IL-10 production (data not shown). Moreover, one of the NSAID aspirin, well known as a NF-{kappa}B inhibitor [33 , 34 ], augmented LPS-induced ROI production (data not shown). These evidences may address the mechanism of distinct, clinical efficacy between macrolides and NSAIDs. For example, aspirin-induced ROI production may cause tissue damage, inducing Ca2+ signaling [35 , 36 ]. The ROI-induced Ca2+ signaling possibly triggers bronchial hyper-responsiveness [25 , 37 ], which may play a causative role in the mechanism of aspirin-induced asthma, termed "aspirin dilemma" in the treatment of chronic respiratory inflammation [38 ], and macrolide treatments are beneficial and effective. Thus, the ROI inhibition as well as the NF-{kappa}B inhibition in inflammatory lesions may play an important role in the beneficial efficacy of the macrolides for the treatment of chronic respiratory inflammation.

Additionally, the increased IL-10 production may contribute to the anti-inflammatory and cytoprotective actions of macrolides. An anti-inflammatory cytokine, IL-10 has recently been reported to induce an antioxidant protein, heme oxygenase-1, which has been implicated in the cytoprotective defense response against oxidative injuries [39 40 41 42 ]. These evidences regarding IL-10 may also explain the advantageous, therapeutic efficacy of macrolides for the treatment of chronic respiratory inflammation.


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CONCLUSION
 
In conclusion, the present study has provided the first experimental evidence for the mechanism of anti-inflammatory actions characterized by NF-{kappa}B inhibition and cytoprotection. Our data also introduce the functional role of the cAMP/CREB pathway in the mechanism of macrolide anti-inflammatory actions.


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ACKNOWLEDGEMENTS
 
This study was supported by research grants from Ministry of Education and Science, Grant-in-Aid #13470324 and #14657627, Japan. K. A. and K-i. K. contributed equally to this work. We thank Dr. Masakazu Hatanaka (Shionogi Institute, Osaka, Japan) for kindly providing HTLV-I–Tax expression vector, Dr. M. Tokudome (Aisei-kai Shounan Hospital, Osumi, Japan) for clinical samples, N. Uto, Y. Soejima, and H. Sameshima for the excellent technical assistance, and K. Yamakuchi-Sakou and T. Soejima for the preparation of this manuscript.

Received March 12, 2003; revised May 24, 2003; accepted June 27, 2003.


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