Yonago Acta medica 2015;58:31–38

Original Article

Effect of Clarithromycin on the Expression of UL16-Binding Protein 2 in
Human Cells
Kensaku Okada,* Hiroki Chikumi,*† Miyako Takata,* Kosuke Yamaguchi,* Haruhiko Makino,* Tsuyoshi Kitaura,*
Masaki Nakamoto,*† Akira Yamasaki,* Tadashi Igishi,† Naoto Burioka‡ and Eiji Shimizu**Division of Medical Oncology and Molecular Respirology, Department of Multidisciplinary Internal Medicine, School of Medicine,
Tottori University Faculty of Medicine, Yonago 683-8504, Japan, †Center for Infectious diseases, Tottori University Hospital, Yonago
683-8504, Japan and ‡Department of Pathological Science and Technology, School of Health Science, Tottori University Faculty of
Medicine, Yonago 683-8503, Japan

ABSTRACT

both A549 and LCSC #2 cells by treatment with clarithromycin. Finally, clarithromycin significantly inhibited the activity of ADAM17 in LCSC #2 cells.
Conclusion These findings suggest that clarithromycin induces ULBP2 expression and reduces the amountof sULBP2, by possibly inhibiting the activity of the potent ULBP2-shedding enzyme ADAM17. Because thesechanges in ULBP2 and sULBP2 levels could activate
NKT cells, this finding might indicate a novel mechanism by which clarithromycin improves the clearance ofP. aeruginosa in chronic respiratory diseases.

Background

Clarithromycin is a macrolide antibiotic
that possesses anti-inflammatory and immunomodulatory properties. Although recent data suggests thatmacrolide antibiotics enhance Pseudomonas aeruginosaclearance from the lung, involving natural killer (NK) T
cells in this process by activating the NKG2D-NKG2D
ligand system, the precise underlying mechanism is still
unclear. In this study, we examined the effect of clarithromycin on a potent NKG2D ligand, UL16-binding
protein 2 (ULBP2), in the lung and its shedding mechanism.
Methods The gene expressions of ULBP2 and theshredder proteinases of ULBP2, a disintegrin and metalloproteinase domain 10 (ADAM10) and ADAM17, were
measured using real-time PCR. The cell surface ULBP2
expression was measured by flow cytometry. The
amount of solubilized ULBP2 (sULBP2) was measured
using an ELISA. The activity of ADAM17 was examined by measurement of fluorescence intensity from thefluorescence resonance energy transfer peptide substratecleaved by ADAM17.
Results Clarithromycin significantly induced transcription of ULBP2 and ADAM17 in both A549 and
LCSC #2 cells, which endogenously express minimal
and abundant levels of ULBP2, respectively. However, there was no significant change on transcriptionof ADAM10. The same tendency was observed when
LCSC #2 cells were treated with tumor necrosis factoralpha processing inhibitor-2 to inhibit ADAM17 activity.
The amount of sULBP2 was significantly decreased in

Key words a disintegrin and metalloprotease domain
17 protein; clarithromycin; Pseudomonas aeruginosa;
UL16 binding protein 2

Clarithromycin is a member of the macrolide antibiotic
class, potent and well-established antimicrobials with
14- and 15-member lactone rings that also possess antiinflammatory and immunomodulatory properties.1–3 The
effect of clarithromycin is mainly attributed to inhibitionof inflammatory cytokine and chemokine production.
The most proverbial demonstrations of the anti-inflammatory and immunomodulatory effects of macrolide
antibiotics were reported in the successful treatment ofdiffuse panbronchiolitis4 and cystic fibrosis.5 These successes expanded the evaluation of macrolide antibiotics
for the treatment of other chronic inflammatory diseases
in the lung.6
Chronic infection of the lung by pathogens such as
Pseudomonas aeruginosa is a life-threatening problem
for patients with diffuse panbronchiolitis,7 cystic fibrosis8 and other chronic inflammatory lung diseases. Recent data suggests that macrolide antibiotics enhanced
the clearance of P. aeruginosa from lung, although they
intrinsically do not have antimicrobial activity for thisbacterium.9 This result is thought to be another beneficial effect of macrolide antibiotics for the treatment oflung diseases; however, the precise mechanism of clarithromycin-induced clearance of P. aeruginosa in thesechronic diseases remains unclear.

Corresponding author: Hiroki Chikumi, MD
chikumi@grape.med.tottori-u.ac.jp.
Received 2014 December 8
Accepted 2015 January 5
Abbreviations: ADAM, a disintegrin and metalloproteinase domain; FRET, fluorescence resonance energy transfer; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; HB-EGF, heparinbinding epidermal growth factor-like growth factor; IL, interleukin; MFI, mean fluorescence intensity; NK, natural killer;
sULBP2, solubilized ULBP2; TAPI-2, TNF-alpha processing inhibitor-2; TGF, transforming growth factor; TNF, tumor necrosis
factor; ULBP2, UL16-binding protein 231

K. Okada et al.
Table 1. Primers used in this study

Recent data also suggests that natural killer (NK) T
cells play a central role in clearing P. aeruginosa fromthe lungs.10 NKT cells are a specialized type of T cellthat share properties of both T cells and NK cells andare emerging as critical regulators of the immune response to infectious agents.11, 12 Additionally, NKT cells
are thought to play a role in managing human infections
such as cystic fibrosis.13, 14 Previous studies showed thatthe activities of NKT cells, CD8+T cells and NK cells
are tightly controlled by the activation receptor NKG2Dthat is expressed on the cell surface of these immune
effector cells.15 The ligands for NKG2D are generally
not expressed in normal cells, but their expression is induced in infected16 or transformed17 cells that should be
eliminated by the host immune system. NKG2D on thesurface of immune effector cells recognizes its ligands
expressed on the surfaces of target cells and subsequently augments the cytolytic activity of the immune effector
cells to promote destruction and clearance of pathogeninfected cells. In line with this, recent data suggests that
expression of NKG2D contributes to the pulmonary
clearance of P. aeruginosa.16, 18 Therefore, this NKG2DNKG2D ligand system is thought to play an essential
role in host immunity during chronic lung infections.
UL16-binding protein 2 (ULBP2) is one of the ligands for NKG2D that is expressed on infected humanlung epithelial cells.19 Recently, we demonstrated thatcell surface ULBP2 is released in soluble form fromthe surface and the resultant soluble ULBP2 (sULBP2)
reduces the cytotoxic activity of human peripheral
blood mononuclear cells.19 Therefore, ULBP2 on thecell surface and sULBP2 may have crucial but opposite
effects on the infection immunity of NKT, CD8+T andNK cells in the lung. However, the effect of clarithromycin on these molecules has not been explored. In thepresent study, we evaluated the effect of clarithromycin
on ULBP2, sULBP2 and their candidate shedder proteinase, a disintegrin and metalloproteinase domain 10(ADAM10) and ADAM17,20 in human cells originatedfrom the lung.

Gene

Ampliconsize(bp)
Sequences of primers

Forward: ccgctaccaagatccttctg
ULBP2 108
		Reverse: ggatgacggtgatgtcatagc
ADAM10 60
Forward: atattacggaacacgagaagctg
		Reverse: tcaatcgctttaacatgactgg
ADAM17 69
Forward: cctttctgcgagagggaac
		Reverse: caccttgcaggagttgtcagt
GAPDH 66
Forward: agccacatcgctgagaca
		Reverse: gcccaatacgaccaaatcc
ADAM, a disintegrin and metalloproteinase domain; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; ULBP2, UL16binding protein 2.

were maintained in RPMI 1640 (Wako) with 10% heatinactivated FBS, 50 units/mL penicillin and 50 units/mL
streptomycin. Both cell lines were cultured in humidified air with 5% CO2 at 37 ˚C.
Reagents

Clarithromycin was obtained from Tokyo Chemical
Industry (Tokyo, Japan). Tumor necrosis factor (TNF)alpha processing inhibitor-2 (TAPI-2) was obtained
from Enzo Life Science (Farmingdale, NY). AntiULBP2 (clone 165903) and recombinant ULBP2-Fc
were obtained from R&D Systems (Minneapolis, MN).
Anti-ULBP2 (BUMO1) was purchased from Bamomab(Munich, Germany) and goat anti-mouse IgG2a was purchased from SouthernBiotech Associates (Birmingham,
AL). Isotype control IgG2a was obtained from SigmaAldrich (St. Louis, MO).
RNA extraction and cDNA synthesis

Total RNA was extracted from cells using a QIAGEN
RNeasyPlus Mini Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. RNA was
dissolved in 50 mL of Rnase-free water and stored at–80 ˚C until use in a cDNA preparation reaction. An
aliquot was removed to determine the concentrationof RNA in a spectrophotometer at 260 nm. For cDNA
synthesis, each 20 mL reaction contained 1 mg of total
RNA, as described in the QIAGEN QuantiTect Reverse
Transcription Kit (Qiagen). Synthesis of cDNA was
performed in a TaKaRa PCR Thermal Cycler (TaKaRa,
Kyoto, Japan) according to the following procedure: theRNA samples were incubated in gDNA Wipeout Buffer(Qiagen) at 42 ˚C for 2 min; Quantiscript Reverse Transcriptase, Quantiscript RT Buffer and RT Primer Mix(Qiagen) were added and incubated at 42 ˚C for 15 min;and the reverse transcriptase was inactivated for 3 min at
95 ˚C. The cDNA was stored at –30 ˚C until use in realtime PCR.

MATERIALS AND METHODS
Cells

A549 lung cancer cell lines were obtained from the
RIKEN cell bank (Tsukuba, Japan). LCSC #2 lung cancer cell lines were provided by the Cell Resource center
for Biomedical Research (Institute of Development,
Aging and Cancer, Tohoku University, Sendai, Japan).
A549 cells were maintained in Dulbecco’s Modified
Eagles Medium (Wako, Osaka, Japan) with 10% heatinactivated fetal bovine serum (FBS), 50 units/mL
penicillin and 50 units/mL streptomycin. LCSC #2 cells
32Effect of clarithromycin on ULBP2Quantitative real-time PCR

Diagnostics) and 4 mL diluted cDNA (1:10). The cycling parameters used were as follows: one denaturation
cycle for 600 s at 95 ˚C and 45 amplification cycles(temperature transition rate of 4.4 ˚C s–1) of 10 s at 95
˚C, annealing for 25 s at 55 ˚C and extension for 1 s at
72 ˚C. Fluorescence readings were taken after each cycle
following the extension step. The LightCycler 480 software generated a standard curve from the standards anddetermined the gene copy number in each test sample.

A LightCycler 480 (Roche Diagnostics, Mannheim,
Germany) was used for all quantitative PCRs. Table
1 shows the gene-specific primers used. All PCR amplification reactions were performed with 0.5 mM
forward primers, 0.5 mM reverse primers (SigmaGenosys, Ishikari, Japan), 0.4 mM hydrolysis probes foreach target gene from a universal probe library (Roche
Diagnostics), 1× LightCycler 480 Probes Master (Roche

Fig. 1. The effect of clarithromycin on the mRNA expression of ULBP2, ADAM10 and ADAM17 in A549 and LCSC #2 cell lines.
A549 and LCSC #2 cells were treated with 0.1, 1 or 10 μg/mL clarithromycin for 24 h after serum starvation and harvested for total RNA
extraction and quantitative real-time PCR. The ratios of gene expression between the target genes (ULBP2, ADAM10 and ADAM17)and internal standard (GAPDH) were expressed relative to those of A549 or LCSC #2 cells without clarithromycin treatment, which are
set at 1.00. Data are expressed as mean values ± SD (n = 3 for each group). *P < 0.05 versus control. A: mRNA expression of ULBP2 in
A549 cells. B: mRNA expression of ULBP2 in LCSC #2 cells. C: mRNA expression of ADAM10 in A549 cells. D: mRNA expressionof ADAM10 in LCSC #2 cells. E: mRNA expression of ADAM17 in A549 cells. F: mRNA expression of ADAM17 in LCSC #2 cells.
ADAM, a disintegrin and metalloproteinase domain; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ULBP2, UL16-binding protein 2.

33

K. Okada et al.

Flow cytometry

The ratios of gene expression between the target genes(ULBP2, ADAM10, and ADAM17) and the internal
standard (GAPDH) were expressed relative to those of
A549 or LCSC #2 without clarithromycin, which is set
at 1.00.

Cells were incubated with monoclonal anti-ULBP2
antibodies or isotype controls. After washing, goatanti-mouse IgG labeled with FITC was added, and thefluorescence intensities of samples were analyzed on
a FACSCalibur (BD Biosciences, La Jolla, CA). Data
from 10,000 cells were collected, and the geometricmean fluorescence intensity (MFI) was calculated using
CELLQuest software (BD Biosciences). MFI ratios were
calculated by dividing the MFI obtained with a specificantibody by that for the isotype control.
ELISA

Two monoclonal anti-ULBP2 antibodies were used todetect sULBP2. Plates were coated with the anti-ULBP2mAb BUMO1 at 2 mg/mL in coating buffer for 24 h at
4 ˚C, then blocked with blocking buffer for 18 h at 4 ˚C.
The blocking buffer was prepared with a commerciallyavailable blocking agent (Applied Bioscience, Mumbai,
India). Next, ULBP2-Fc (standard) and the samples wereadded, and the plates were incubated for 1 h at room
temperature. For analysis of serum samples, ULBP2-Fc
was diluted in blocking buffer with 25% horse serum,and sera were diluted 1:3 in blocking buffer before addition to the plates. After incubation, the detector mAb(anti-ULBP2, clone 165903) was added at 0.5 mg/mL
in blocking buffer for 1 h at room temperature, followed
by incubation with anti-mouse IgG2a-HRP (1:10,000
in blocking buffer) for 1 h at 37 ˚C. Color was developed with a Sumilon peroxidase ELISA kit (Sumitomo
Chemical, Tokyo), and the absorbance was read at 490
nm on a Model 680 microplate reader (Bio-Rad Laboratories, Hercules, CA).
TACE activity assay

The activity of ADAM17, which is also known as
TACE, was determined using the SensoLyte 520 TACE
Activity Assay Kit (AnaSpec, San Jose, CA) with 10µg of cell lysate proteins from each sample according
to the manufacturer’s protocol. This assay uses a 5-carboxyfluorescein (5-FAM)-labeled fluorescence resonance energy transfer (FRET) peptide substrate. Upon
cleavage of the FRET peptide by the active enzyme, thefluorescence of 5-FAM is recovered and can be monitored at excitation/emission wavelengths of 490/520 nm.
Fluorescence of the cleavage product was measured ina fluorescence microplate reader (TECAN, Seestrasse,
Switzerland). Results are expressed as the mean change
in fluorescence intensity per min.

Fig. 2. The effect of clarithromycin on the cell surface expressionof ULBP2 in A549 and LCSC #2 cell lines. A549 and LCSC #2
cells were treated with or without 10 μg/mL clarithromycin or
50 μM TAPI-2 for 24 h after serum starvation and harvested forflow cytometry. A: A549 cells treated with or without 10 μg/mL
clarithromycin. B: LCSC #2 cells treated with or without 10 μg/
mL clarithromycin. C: A549 cells treated with or without 50 μM
TAPI-2. D: LCSC #2 cells treated with or without 50 μM TAPI2. TAPI-2, TNF-alpha processing inhibitor-2; ULBP2, UL16binding protein 2.

Statistical analysis

Statistical analyses were performed using the SPSS 21.0
software (International Business Machines Corporation,
34

Effect of clarithromycin on ULBP2Armonk, NY) and P < 0.05 was regarded as statisticallysignificant.

10 µg/mL clarithromycin for 24 h. These data suggestthat clarithromycin induces transcription of ULBP2 and
ADAM17.

RESULTS
Effect of clarithromycin on ULBP2 mRNA expression

Effect of clarithromycin on cell surface and soluble
ULBP2

To evaluate the influence of clarithromycin on ULBP2
and its shedding mechanism, we first assessed the effect of clarithromycin on mRNA expression of ULBP2,
ADAM10 and ADAM17. We used two cell lines, A549
and LCSC #2, which express ULBP2 at low and highlevels, respectively. Transcript levels of ULBP2 weresignificantly up-regulated in A549 (Fig. 1A) and LCSC
#2 (Fig. 1B) cells treated with 10 µg/mL clarithromycin
for 24 h. Additionally, although there was no significant
effect of clarithromycin on the mRNA expression of
ADAM10 in A549 (Fig. 1C) or LCSC #2 (Fig. 1D) cells,
ADAM17 mRNA expression was up-regulated in A549
(Fig. 1E) and LCSC #2 (Fig. 1F) cells treated with 1 or

Next, to identify the effect of clarithromycin on protein expression of ULBP2, we evaluated the changesof cell surface ULBP2 expression in A549 and LCSC
#2 cells after 24 h treatment with or without 10 µg/mL
clarithromycin (Figs. 2A and B). Although there was no
significant difference of cell surface ULBP2 expressionin A549 cells with or without 10 µg/mL clarithromycin
treatment (Fig. 2A, MFI ratio: 1.13 versus 1.11, respectively), the expression level of ULBP2 in LCSC #2 cellswith 10 µg/mL clarithromycin was significantly higher
than that without clarithromycin (Fig. 2B, MFI ratio:
22.6 versus 11.5, respectively). To test the contribution of
ULBP2 shedding to this change, we next examined theeffect of TAPI-2, an ADAM17 inhibitor, on the expression of ULBP2. The same tendency was observed when
LCSC #2 cells were treated with 50 µM TAPI-2 for 24
h (Figs. 2C and D). Therefore, clarithromycin may mimic the effect of TAPI-2, especially for cells with abundant expression of ULBP2 such as LCSC #2 cells. These
data suggest that clarithromycin induces cell surface
ULBP2 expression. In addition, the mechanism of thiseffect is presumably induction of ULBP2 gene transcription and inhibition of ULBP2 shedding by ADAM17.
Quantification of sULBP2Previously, we showed that ULBP2 is shed into the culture medium in proportion to the level of its cell surface
expression by non-small cell lung cancer cell lines.19
Therefore, to reveal the effect of clarithromycin on
sULBP2, we used ELISA to analyze culture supernatants from A549 (Fig. 3A) and LCSC #2 cells (Fig. 3B)
at 24 and 72 h after treatment with or without clarithromycin. The number of the cells did not differ significantly under each experimental condition (data not shown);
however, the amount of sULBP2 increased over time inthese cell lines. The amount of sULBP2 with clarithromycin was significantly decreased relative to that without clarithromycin in both A549 and LCSC #2 cells (Figs.
3A and B). These findings suggest an inhibitory effect ofclarithromycin on the ULBP2 shedding mechanism.

Fig. 3. The effect of clarithromycin on the level of sULBP2 in
A549 and LCSC #2 cell lines. Measurement of sULBP2 in culture
supernatants using ELISA. A549 and LCSC #2 cells were treatedwith or without 10 μg/mL clarithromycin for 24 or 72 h after
serum starvation. After treatment, the culture supernatants were
harvested for ELISA. Data are expressed as mean values ± SD (n= 3 for each group). *P < 0.05. A: A549 cells treated with or without 10 μg/mL clarithromycin. B: LCSC #2 cells treated with orwithout 10 μg/mL clarithromycin. sULBP2, solubilized ULBP2.

Evaluation of ADAM17 activity
To explore the effect of clarithromycin on the ULBP2
shedding mechanism, we next examined the effect ofclarithromycin on the activity of ADAM17 because reduction of sULBP2 was observed despite increased transcription of the shedder enzyme ADAM17. As shown in35

K. Okada et al.

transcription of mucin,22 IL-823 and various cytokines
such as TNF-alpha, interferon gamma, interleukin (IL)-2,
IL-4, IL-5 and IL-6.24 On the other hand, transcriptionof IL-1025 and alpha1-acid glycoprotein was induced byclarithromycin in hepatocytes.26 Toward understanding the mechanism of gene transcription modulation byclarithromycin, a previous study reported that clarithromycin inhibits activator protein-1 transcription factor23
or potentiates glucocorticoid response element.26 The
precise mechanism leading to up-regulation of ULBP2and ADAM17 is still unclear, but it may occur throughthe modulation of certain transcription factors. Because
most previous reports focused on the effect of clarithromycin on the inflammatory cytokines, its effect on otherproteins involved in the immune process and its mechanism of action must be explored in future studies.
In addition to its role in transcriptional regulation,
we revealed that clarithromycin inhibits the enzymatic
activity of ADAM17. The inhibitory effect of clarithromycin on enzyme activity has been reported inother enzymes such as cytochrome P450 3A,27 humanserum paraoxonase28 and glycosidase.29 This effect ofclarithromycin on ADAM17 activity represents a newmechanism that may explain the recent reports of broad
pleiotropic activities of macrolide antibiotics for immunomodulatory,30 anti-inflammatory31 and antitumor32 effects. Previous studies revealed that ADAM17, originallyidentified as an enzyme that proteolytically cleaves proTNF-alpha to generate soluble TNF-alpha,33 cleaves andsolubilizes a wide range of membrane-bound proteins
including transforming growth factor (TGF)-alpha34 andheparin-binding epidermal growth factor-like growth
factor (HB-EGF).35 The substrate proteins of ADAM17
make obvious contributions to various pathogeneses, forexample, TNF-alpha is involved in inflammation, andboth TGF-alpha and HB-EGF contribute to tumorigenesis. Therefore, inhibition of the activity of ADAM17 byclarithromycin might represent a novel mechanism forits effect on these pathological conditions.
Recent reports suggested that P. aeruginosa infection induced the surface expression of human NKG2D
ligands such as ULBP2 in airway epithelial cells andsignified the indispensability of induced NKG2Dmediated immune activation in the clearance of thisbacterium.16, 18 In addition, we previously revealed that P.
aeruginosa is actively involved in this process by secreting a quorum sensing molecule, N-3-oxo-dodecanoyl
homoserine lactone, that decreases cell surface ULBP2
and increases sULBP2.36 This intervention by P. aeruginosa leads to attenuation of the host NKG2D-mediatedimmune response and is beneficial for its survival in
the human lung. Our novel finding in this study that

Fig. 4. The effect of clarithromycin on ADAM17 activity in A549
and LCSC #2 cell lines. ADAM17 activity was determined using
the SensoLyte 520 TACE Activity Assay Kit with 10 µg of celllysate proteins for each sample according to the manufacturer’s
protocol. A549 and LCSC #2 cells were treated with or without
10 μg/mL clarithromycin for 24 h after serum starvation andharvested for the assay. Data are expressed as the mean change influorescence intensity per min ± SD (n = 3 for each group). *P <0.05. ADAM, a disintegrin and metalloproteinase domain.

Fig. 4, 10 µg/mL clarithromycin significantly inhibitedthe activity of ADAM17 in LCSC #2 cells with highcell surface ULBP2 expression. However, the effect ofclarithromycin on A549 cells, which expressed lower
levels of ULBP2, was not significant. This finding demonstrates that clarithromycin inhibits ADAM17 activity
and, consequently, the ULBP2 shedding mechanism.
DISCUSSION
In this study, we examined the effect of clarithromycin
on ULBP2 expression. Our results showed that clarithromycin induced the transcription and expression of cellsurface ULBP2 while reducing the amount of sULBP2
in the culture medium. Additionally, clarithromycin inhibited the activity of ADAM17, a shedder enzyme forcell surface NKG2D ligands.21 Because immune effector
cells such as NKT cells, CD8+T cells and NK cells areactivated by enhanced expression of ULBP2 on the surfaces of target cells and are affected by only low levels
of sULBP2, these pleiotropic effects of clarithromycin
on ULBP2 expression might contribute to the enhanced
function of immune effector cells, thereby augmenting P.
aeruginosa clearance in the lung.
In this study, we first reported the up-regulation of
ULBP2 and ADAM17 gene transcription in human cells
derived from the lung. Previous studies reported thatclarithromycin influences the gene expression of various
proteins in human cells, including both reduction and induction of genes involved in inflammation. For example,
studies have shown that clarithromycin inhibited gene36Effect of clarithromycin on ULBP2clarithromycin increases the cell surface ULBP2 expression and decreases sULBP2 suggests a mechanism bywhich it counteracts the immunosuppressive activity of P.
aeruginosa and might explain the effectiveness of clarithromycin in treating chronic P. aeruginosa infection inchronic lung diseases.37
In conclusion, we revealed that clarithromycin induces expression of ULBP2 and, through ADAM17, reduces sULBP2. These findings suggest the possibilities
that clarithromycin can activate NKT cells and decrease
the colonization of bacteria in patients with chronic
respiratory diseases such as diffuse panbronchiolitis.
Therefore, further study toward augmenting the effectof clarithromycin on NKT cell activity through ULBP2
might be useful for developing new macrolide-based
treatments for chronic respiratory diseases.

crobiol. 2007;5:405-17. PMID: 17487145.
12 Matsuda JL, Mallevaey T, Scott-Browne J, Gapin L. CD1drestricted iNKT cells, the ‘Swiss-Army knife’ of the immune system. Curr Opin Immunol. 2008;20:358-68. PMID:
18501573.
13 Silk JD, Salio M, Brown J, Jones EY, Cerundolo V. Structuraland functional aspects of lipid binding by CD1 molecules.
Annu Rev Cell Dev Biol. 2008;24:369-95. PMID: 18593354.
14 Rzemieniak SE, Hirschfeld AF, Victor RE, Chilvers MA,
Zheng D, van den Elzen P, et al. Acidification-dependent activation of CD1d-restricted natural killer T cells is intact in cystic fibrosis. Immunology. 2010;130:288-95. PMID: 20102408.
15 Gonzalez S, Lopez-Soto A, Suarez-Alvarez B, Lopez-Vazquez
A, Lopez-Larrea C. NKG2D ligands: key targets of the immune response. Trends Immunol. 2008;29:397-403. PMID:
18602338.
16 Borchers MT, Harris NL, Wesselkamper SC, Zhang S, Chen
Y, Young L, et al. The NKG2D-activating receptor mediates
pulmonary clearance of Pseudomonas aeruginosa. Infect Immun. 2006;74:2578-86. PMID: 16622193.
17 Bauer S, Groh V, Wu J, Steinle A, Phillips JH, Lanier LL, et
al. Activation of NK cells and T cells by NKG2D, a receptor
for stress-inducible MICA. Science. 1999;285:727-9. PMID:
10426993.
18 Wesselkamper SC, Eppert BL, Motz GT, Lau GW, Hassett
DJ, Borchers MT. NKG2D is critical for NK cell activation
in host defense against Pseudomonas aeruginosa respiratory
infection. J Immunol. 2008;181:5481-9. PMID: 18832705.
19 Yamaguchi K, Chikumi H, Shimizu A, Takata M, Kinoshita
N, Hashimoto K, et al. Diagnostic and prognostic impact ofserum-soluble UL16-binding protein 2 in lung cancer patients.
Cancer Sci. 2012;103:1405-13. PMID: 22587355.
20 Wolpert F, Tritschler I, Steinle A, Weller M, Eisele G. A disintegrin and metalloproteinases 10 and 17 modulate the immunogenicity of glioblastoma-initiating cells. Neuro Oncol.
2014;16:382-91. PMID: 24327582.
21 Waldhauer I, Goehlsdorf D, Gieseke F, Weinschenk T,
Wittenbrink M, Ludwig A, et al. Tumor-associated MICA
is shed by ADAM proteases. Cancer Res. 2008;68:6368-76.
PMID: 18676862.
22 Kaneko Y, Yanagihara K, Seki M, Kuroki M, Miyazaki Y,
Hirakata Y, et al. Clarithromycin inhibits overproduction of
muc5ac core protein in murine model of diffuse panbronchiolitis. Am J Physiol Lung Cell Mol Physiol. 2003;285:L84753. PMID: 12818892.
23. Abe S, Nakamura H, Inoue S, Takeda H, Saito H, Kato S, etal. Interleukin-8 gene repression by clarithromycin is mediated by the activator protein-1 binding site in human bronchial
epithelial cells. Am J Respir Cell Mol Biol. 2000;22:51-60.
PMID: 10615065.
24 Morikawa K, Zhang J, Nonaka M, Morikawa S. Modulatory effect of macrolide antibiotics on the Th1- and Th2-type
cytokine production. Int J Antimicrob Agents. 2002;19:53-9.
PMID: 11814768.
25 Umezawa M, Tanaka N, Takeda K, Ihara T, Sugamata
M. Clarithromycin and telithromycin increases interleukin-10 expression in the rat endometriosis model. Cytokine.
2011;55:339-42. PMID: 21665488.
26 Komori T, Kai H, Shimoishi K, Kabu K, Nonaka A,
Maruyama T, et al. Up-regulation by clarithromycin ofalpha(1)-acid glycoprotein expression in liver and primarycultured hepatocytes. Biochem Pharmacol. 2001;62:1391-7.
PMID: 11709199.

The authors declare no conflict of interest.
REFERENCES
1 Culic O, Erakovic V, Parnham MJ. Anti-inflammatory effects
of macrolide antibiotics. Eur J Pharmacol. 2001;429:209-29.
PMID: 11698042.
2 Lopez-Boado YS, Rubin BK. Macrolides as immunomodulatory medications for the therapy of chronic lung diseases. Curr
Opin Pharmacol. 2008;8:286-91. PMID: 18339582.
3 Shinkai M, Henke MO, Rubin BK. Macrolide antibiotics as
immunomodulatory medications: proposed mechanisms ofaction. Pharmacol Ther. 2008;117:393-405. PMID: 18289694.
4 Kudoh S, Azuma A, Yamamoto M, Izumi T, Ando M. Improvement of survival in patients with diffuse panbronchiolitis
treated with low-dose erythromycin. Am J Respir Crit CareMed. 1998;157(6 Pt 1):1829-32. PMID: 9620913.
5 Clement A, Tamalet A, Leroux E, Ravilly S, Fauroux B, Jais
JP. Long term effects of azithromycin in patients with cystic
fibrosis: A double blind, placebo controlled trial. Thorax.
2006;61:895-902. PMID: 16809416.
6 Basyigit I, Yildiz F, Ozkara SK, Yildirim E, Boyaci H, Ilgazli
A. The effect of clarithromycin on inflammatory markers inchronic obstructive pulmonary disease: preliminary data. Ann
Pharmacother. 2004;38:1400-5. PMID: 15252191.
7 Homma H, Yamanaka A, Tanimoto S, Tamura M, Chijimatsu
Y, Kira S, et al. Diffuse panbronchiolitis. A disease of thetransitional zone of the lung. Chest. 1983;83:63-9. PMID:
6848335.
8 Emerson J, Rosenfeld M, McNamara S, Ramsey B, Gibson
RL. Pseudomonas aeruginosa and other predictors of mortality and morbidity in young children with cystic fibrosis. Pediatr Pulmonol. 2002;34:91-100. PMID: 12112774.
9 Yanagihara K, Tomono K, Imamura Y, Kaneko Y, Kuroki M,
Sawai T, et al. Effect of clarithromycin on chronic respiratory
infection caused by Pseudomonas aeruginosa with biofilm
formation in an experimental murine model. J Antimicrob
Chemother. 2002;49:867-70. PMID: 12003986.
10 Nieuwenhuis EE, Matsumoto T, Exley M, Schleipman RA,
Glickman J, Bailey DT, et al. CD1d-dependent macrophagemediated clearance of Pseudomonas aeruginosa from lung.
Nat Med. 2002;8:588-93. PMID: 12042809.
11 Tupin E, Kinjo Y, Kronenberg M. The unique role of naturalkiller T cells in the response to microorganisms. Nat Rev Mi-37

K. Okada et al.
27 Quinney SK, Malireddy SR, Vuppalanchi R, Hamman MA,
Chalasani N, Gorski JC, et al. Rate of onset of inhibition ofgut-wall and hepatic CYP3A by clarithromycin. Eur J Clin
Pharmacol. 2013;69:439-48. PMID: 22777148.
28 Sinan S, Kockar F, Gencer N, Yildirim H, Arslan O. Amphenicol and macrolide derived antibiotics inhibit paraoxonase enzyme activity in human serum and human hepatoma
cells (HepG2) in vitro. Biochemistry (Mosc). 2006;71:46-50.
PMID: 16457617.
29 Sadeghi-Khomami A, Lumsden MD, Jakeman DL. Glycosidase inhibition by macrolide antibiotics elucidated by STDNMR spectroscopy. Chem Biol. 2008;15:739-49. PMID:
18635010.
30 Giamarellos-Bourboulis EJ. Macrolides beyond the conventional antimicrobials: a class of potent immunomodulators. Int
J Antimicrob Agents. 2008;31:12-20. PMID: 17935949.
31 Zalewska-Kaszubska J, Gorska D. Anti-inflammatory capabilities of macrolides. Pharmacol Res. 2001;44:451-4. PMID:
11735349.
32 Wang L, Kitaichi K, Hui CS, Takagi K, Sakai M, Yokogawa
K, et al. Reversal of anticancer drug resistance by macrolide
antibiotics in vitro and in vivo. Clin Exp Pharmacol Physiol.
2000;27:587-93. PMID: 10901387.

33 McGeehan GM, Becherer JD, Bast RC Jr, Boyer CM,
Champion B, Connolly KM, et al. Regulation of tumour necrosis factor-alpha processing by a metalloproteinase inhibitor.
Nature. 1994;370:558-61. PMID: 8052311.
34 Peschon JJ, Slack JL, Reddy P, Stocking KL, Sunnarborg SW,
Lee DC, et al. An essential role for ectodomain shedding in
mammalian development. Science. 1998;282:1281-4. PMID:
9812885.
35 Jackson LF, Qiu TH, Sunnarborg SW, Chang A, Zhang C,
Patterson C, et al. Defective valvulogenesis in HB-EGF and
TACE-null mice is associated with aberrant BMP signaling.
EMBO J. 2003;22:2704-16. PMID: 12773386.
36 Matsunaga S, Chikumi H. Pseudomonas aeruginosa quorumsensing molecule N-3-oxo-dodecanoyl homoserine lactone
down-regulate cell surface ULBP2 on lung cancer cell lines.
Yonago Igaku Zasshi. 2011;62:77-90. Japanese.
37 Tateda K, Ishii Y, Kimura S, Horikawa M, Miyairi S,
Yamaguchi K. Suppression of Pseudomonas aeruginosa
quorum-sensing systems by macrolides: a promising strategy
or an oriental mystery? J Infect Chemother. 2007;13:357-67.
PMID: 18095083.

38