1Department of Respiratory Oncology and Molecular Medicine, Division of Cancer Control, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575; 2Sendai Kousei Hospital, 4-15 Hirose-chou, Aoba-ku, Sendai 980-0873, Japan
Received 12 October 2001; returned 25 November 2001; revised 2 January 2002; accepted 24 January 2002.
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Abstract |
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Introduction |
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Interleukin-8 (IL-8) is a member of the CXC chemokines and is mainly produced by activated monocytes. In Gram-negative infections, lipopolysaccharide (LPS) activates monocytes.12,13 Monocytes, in turn, produce IL-8 and thus attract inflammatory cells to the site of infection.1317 It has been reported that IL-8 is the chemokine responsible for the maintenance of chronic inflammation in DBP,3,5,8,18,19 in gastritis caused by Helicobacter pylori,2022 and in other chronic inflammatory diseases.2325 Although several studies have re-ported that macrolides suppress IL-8 production by bronchial epithelial cells stimulated by pro-inflammatory cytokines,57 epithelial cells do not produce IL-8 in response to LPS, whereas monocytes do.26 Therefore, to investigate the effect of macrolides in DPB, studies on monocytes are mandatory. Moreover, as monocytes are involved in all chronic inflammation, such studies should provide valuable information to probe the utility of macrolides for the treatment of other chronic inflammatory diseases. Nevertheless, the effect of macrolides on LPS-stimulated monocytes has not been reported.
Understanding the biochemical mechanism by which macrolides affect IL-8 production is important. FK 506 and glucocorticoids both have anti-inflammatory and immunosuppressive activities, and inhibit the IL-8 promoter through activator protein-1 (AP-1) or nuclear factor-B (NF-
B).2729 Macrolides suppress the IL-8 promoter through AP-1 and NF-
B in bronchial epithelial cells.30,31 Recently, LPS has been shown to stimulate the Toll-like receptor 4, which eventually activates AP-1 and NF-
B.32 Therefore, AP-1 and NF-
B play major roles in the cellular reaction in inflammation. The effects of macrolides on AP-1 and NF-
B in LPS-stimulated monocytes need to be explored.
Here we report that clarithromycin decreases IL-8 production by LPS-stimulated monocytes and suppresses IL-8 mRNA expression through NF-B and AP-1.
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Materials and methods |
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LPS, benzamidine, aprotinin, leupeptin, phenylmethylsulphonyl fluoride (PMSF) and RPMI 1640 medium were purchased from Sigma Chemical Co. (St Louis, MO, USA). RPMI 1640 medium and fetal calf serum (FCS; Gibco-BRL, Life Technologies, Grand Island, NY, USA) were confirmed to contain <0.01 EU/mL endotoxin by the Limulus amoebocyte lysate test (Bio Whittaker Inc., Walkersville, MD, USA). Clarithromycin (Taisho Pharmaceutical Co., Tokyo, Japan) was dissolved in ethanol to make a stock solution (2 mg/mL).
Human peripheral blood monocytes and a human acute monocytic leukaemia cell line, THP-1
Peripheral blood was drawn from healthy volunteers. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation on a Ficollsodium diatrizoate solution (Ficoll-Paque; Pharmacia Biotech, Uppsala, Sweden) and were sus-pended in RPMI 1640 medium supplemented with 10% heat-inactivated FCS. PBMCs were then plated on FCS-coated plates and incubated for 1 h at 37°C in a humidified 95% air/5% CO2 atmosphere. Non-adherent cells were removed by washing with PBS. More than 90% of the adherent cells were morphologically identified as monocytes. A human acute monocytic leukaemia cell line, THP-1, was obtained from the Cell Resource Centre for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, and was maintained in RPMI 1640 medium with 10% heat-inactivated FCS without antibiotics at 37°C under a 95% air/5% CO2 atmosphere.
Bacterial strains and isolation of bacterial lysates
Escherichia coli was isolated from a clinical sample in this laboratory. P. aeruginosa PAO1 strain was obtained from the American Type Culture Collection (no. 39018). A bacterial lysate of H. pylori was purchased from Biodesign International (Saco, ME, USA). E. coli was cultured overnight in LuriaBertani broth at 37°C and P. aeruginosa was cultured in MuellerHinton broth. Cells were collected by centrifugation at 2000g for 10 min. The cells were lysed in 300 µL of B-PER Reagent (Pierce, Rockford, IL, USA), centrifuged at 6000g for 5 min, and the supernatant (lysate) was kept at 80°C.
Plasmids
A series of plasmids containing a serially deleted IL-8 pro-moter fragment [pIL-8(1481) Luc, pIL-8(391) Luc, pIL-8 (335) Luc, pIL-8(130) Luc, pIL-8(112) Luc and promoterless luciferase plasmid pIL-8(0)]33 was generously provided by Dr H. Nakamura (Yamagata University School of Medicine). An NF-B reporter plasmid, ELAM-Luc,34 was a gift from Dr M. Fenton (Boston University School of Medicine, Boston, MA, USA). An enhancerless promoter fragment of the cytomegalovirus (CMV) immediate early gene (GenBank accession no. X03922: nt 10661148) was isolated from pIRES (Clontech, Palo Alto, CA, USA) and placed upstream of the luciferase gene in pGL3-Basic (Promega, Madison, WI, USA) to make pMiniCMV-luc. Either the AP-1 binding site (130 to 107) or the NF-
B binding site (106 to 65), or both binding sites from the IL-8 promoter, were placed upstream of the mini-CMV promoter in pMiniCMV-luc to make p(AP-1)-MiniCMV-luc, p(NF-
B)-MiniCMV-luc or p(AP-1)-(NF-
B)-MiniCMV-luc. An AP-1 reporter plasmid containing multi-ple copies of a typical AP-1 binding sequence (pAP-1-Luc), an NF-
B reporter plasmid containing multiple copies of a typical NF-
B binding sequence (pNF-
B-Luc) and a negative control vector without any binding sequences (pLuc-MCS) were purchased from Stratagene (La Jolla, CA, USA).
Measurement of IL-8 levels in culture supernatants
Human peripheral monocytes or THP-1 cells were incubated in a culture medium containing a bacterial lysate (final con-centration of bacterial protein = 1 mg/L) or LPS (1 mg/L) with or without clarithromycin (10 mg/L). After 6 h, the IL-8 pro-tein concentration in the culture medium was measured by an IL-8-specific, sandwich enzyme-linked immunosorbent assay (ELISA; BioSource International Inc., Camarillo, CA, USA).
DNA transfection and luciferase assay
Individual plasmids, together with a Renilla luciferase expression plasmid [pRL-TK: an internal control to indicate the total cellular transcription level (Promega)], were co-transfected into THP-1 cells using the Effectene Transfection Reagent (Qiagen GmbH, Hilden, Germany). After 48 h, the cells were plated into a 96-well plate (8 x 105 cells per well) and exposed to LPS (0 or 1 mg/L) and clarithromycin (0 or 10 mg/L) in RPMI 1640 medium with 10% FCS. After 4 h, cells were harvested and the luciferase (i.e. firefly luciferase) activity and the Renilla luciferase activity were measured using the Dual-Luciferase reporter assay system (Promega). The luciferase activity was normalized by the Renilla luciferase activity to calculate the relative luciferase activity.
Extraction of nuclear proteins
THP-1 cells (2 x 107) were incubated with LPS (0 or 1 mg/L) and clarithromycin (0 or 1 mg/L) for 4 h. Cells were washed twice with PBS and were collected by centrifugation at 500g at 4°C for 5 min. After lysing the cell membranes by the cytoplasmic extraction reagent (Pierce) containing 0.5 mg/mL benzamidine, 2 mg/L aprotinin, 2 mg/L leupeptin and 0.75 mM PMSF, the cell nuclei were collected by centrifugation at 5000g at 4°C for 5 min. Nuclear membranes were lysed in nuclear extraction reagent (Pierce) containing 0.5 mg/mL benzamidine, 2 mg/L aprotinin, 2 mg/L leupeptin and 2 mM PMSF, and centrifuged at 5000g at 4°C for 10 min. The supernatant (nuclear extract) was aliquoted and kept at 80°C. Protein concentration was determined using the BCA protein assay reagent (Pierce).
Electromobility shift assay (EMSA)
EMSA was carried out using the Gel shift assay system (Pro-mega). The AP-1 probe (5'-AGTGTGATGACTCAGGTTTGCCC-3') and the NF-B probe (5'-GCAAATCGTGGGAATTTCCTCTGACA-3') have the putative AP-1 or NF-
B binding sequences from the IL-8 promoter (underlined). Since the putative NF-
B binding sequence is not a typical binding sequence, we tested the consensus NF-
B probe (5'-GATCCAGGGGACTTTCCCTAGC-3') as well. The probes were end-labelled with [
-32P]adenosine triphosphate using T4 polynucleotide kinase and were purified by a G-25 spin column (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). Nuclear extracts containing 6 µg of protein were incubated in a reaction mixture containing 10 mM TrisHCl (pH 7.5), 50 mM NaCl, 0.5 mM EDTA, 1 mM MgCl2, 0.5 mM DTT, 4% glycerol and 0.05 mg/mL poly(deoxyinosine-deoxycytosine) for 10 min, and subsequently incubated with the same buffer containing 35 fmol of the labelled probe at room temperature for 20 min. In the competition experiments, 1.75 pmol of the unlabelled probe or of the unlabelled mutant probe were added together with the labelled oligo. Mutant probes used were the mutant AP-1 (5'-AGTGTGATGACTtgGGTTTGCCC-3'), the mutant NF-
B (5'-GCAAATCGT-GGcttTTTCCTCTGACA-3'; IL-8 promoter specific) and mutant consensus NF-
B (5'-GATCCAGGGcACTTTCCCTAGC-3'; consensus). In the inhibition experiments, nuclear extracts were preincubated with 1 µL of antibodies against human c-Fos, c-Jun, Jun B, Jun D (for AP-1), NF-
B p50 or NF-
B p65 (for NF-
B) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) before adding the radiolabelled AP-1 or NF-
B probe. Samples were electrophoresed in a 6% DNA retardation gel (Invitrogen Co., Carlsbad, CA, USA) in 0.5x Tris borateEDTA (TBE) buffer at 300 V for 30 min. The gel was dried and subjected to autoradiography.
Statistical analysis
Data are expressed as means ± S.D. The significant difference between the means was tested by Students two-tailed t test. A P value of <0.05 was considered statistically significant.
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Results |
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Clarithromycin suppresses the IL-1- or tumour necrosis factor- (TNF-
) induced IL-8 mRNA expression in human bronchial epithelial cells,7,30 indicating that the IL-8 promoter is one of the main targets of clarithromycin. We investigated the effect of LPS and clarithromycin on the IL-8 promoter using IL-8 promoter-luciferase constructs transfected into THP-1 cells. LPS increased the IL-8 promoter activity, and clarithromycin antagonized LPS (see pNAF in Figure 2). This is consistent with the result shown in Figure 1, and shows that LPS and clarithromycin affected the production of IL-8 at the mRNA transcription level. Experiments using serial deletion mutants showed the following: (i) the presence of the NF-
B binding sequence was necessary for LPS and clarithromycin responsiveness of the promoter (see pN112 in Figure 2); (ii) the presence of the AP-1 binding sequence enhanced both LPS and clarithromycin responsiveness (pN130 in Figure 2); and (iii) the sequence upstream of 130 altered the magnitude of the responses without changing the proportions among the responses (pN335 and pN391 in Figure 2), indicating the presence of the enhancer sequences. We conclude from these data that the AP-1 and NF-
B binding sequences are the key elements responsible for the LPS and clarithromycin responsiveness of the IL-8 promoter.
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Nuclear extract from the untreated THP-1 cells showed a weak binding to the AP-1 probe (Figure 4a). LPS treatment significantly increased the binding. The binding was inhibited by an excess of the unlabelled AP-1 probe, but not by the un-labelled mutant AP-1 probe. Clarithromycin suppressed the binding induced by LPS. This result shows that the binding was specific to the AP-1 probe, and the binding activity parallels the luciferase activity in Figure 3. Similar results were obtained in the experiments using the NF-B probe, or the consensus NF-
B probe (Figure 4b and c).
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Discussion |
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To investigate other diseases in which the anti-inflammatory effect of macrolides might be beneficial, it is important to identify the types of cells on which macrolides exert this effect. In Gram-negative infections, monocytes produce IL-8 in response to LPS, whereas epithelial cells do not.26 Epi-thelial cells secondarily produce IL-8 in response to IL-1, IL-1ß or TNF-
, which are the products of LPS-stimulated monocytes. Therefore, if macrolides do not act on LPS-stimulated monocytes, they are unlikely to exert the anti-inflammatory effect in such infections. Nevertheless, information on the effects of macrolides on LPS-stimulated monocytes has not been available. We observed that most of the DNA transfection methods currently available are incapable of transiently introducing DNA into monocytes or into the monocytic cell line THP-1 at a satisfactory efficiency, and such difficulty may hinder the experiments. A transfection reagent that has recently become available enabled us and others to use THP-1 cells (http://www.qiagen.com/transfectiontools/transquest/index.asp). Our results, together with those previously reported on epithelial cells,30 show that clarithromycin acts on both monocytes and epithelial cells, indicating that clarithromycin may efficiently suppress IL-8 production at the sites of infection.
Our results present a hypothesis that macrolides may be effective for more chronic inflammatory diseases than described above, especially in those where Gram-negative bacteria are involved, such as H. pylori-associated chronic gastritis. Several studies have demonstrated that LPS from H. pylori stimulates gastric epithelial cells to produce IL-8, which in turn elicits chronic gastritis.20,22,27 Amoxicillin, tetracycline, metronidazole and macrolides (mainly clarithromycin) in combination with proton pump inhibitors or bismuth salts comprise the standard therapy.36,37 Our results indicate that clarithromycin may ameliorate inflammation caused by LPS from H. pylori through its anti-inflammatory activity. Although clarithromycin-resistant H. pylori strains have been reported, clarithromycin may still be beneficial owing to its anti-inflammatory activity.
We have shown that clarithromycin suppresses the LPS signalling that leads to both AP-1 and NF-B. Others have shown that clarithromycin suppresses TNF-
and IL-1ß signalling leading to both AP-1 and NF-
B.31 These results indicate that clarithromycin may act in the pathway(s) after the point where the pathway from LPS and the pathway from TNF-
and IL-1ß converge.
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Acknowledgements |
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Footnotes |
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References |
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