Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 May;44(5):606-13.
doi: 10.1165/rcmb.2010-0384OC. Epub 2011 Jan 14.

Up-regulation of MUC18 in airway epithelial cells by IL-13: implications in bacterial adherence

Glenn C Simon  1 , Richard J Martin, Sean Smith, Jyoti Thaikoottathil, Russell P Bowler, Stephen J Barenkamp, Hong Wei Chu
Affiliations

Up-regulation of MUC18 in airway epithelial cells by IL-13: implications in bacterial adherence

Glenn C Simon et al. Am J Respir Cell Mol Biol. 2011 May.

Abstract

Airway bacterial infections are a major problem in lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. Increased Th2 cytokines, such as IL-13, are observed in lung diseases and may contribute to bacterial infections. How Th2 cytokines affect bacterial infection remains unknown. MUC18, an adhesion molecule shown to be involved in the pathogenesis of malignant melanoma, has been recently identified in airway epithelial cells of patients with COPD. We investigated MUC18 regulation by IL-13 and the role of MUC18 in bacterial adherence to epithelial cells. Human airway tissues, brushed bronchial epithelial cells from normal subjects and subjects with asthma, and epithelial cell lines (e.g., HEK293 cells) were used to study the regulation of MUC18 by IL-13 and the involvement of MUC18 in bacterial (e.g., Mycoplasma pneumoniae [Mp] and nontypeable Haemophilus influenzae [NTHi]) adherence to epithelial cells. Asthmatic bronchial epithelium expressed higher levels of MUC18 than normal bronchial epithelium. IL-13 increased MUC18 in cultured bronchial epithelial cells from normal subjects and particularly from subjects with asthma. IL-13-induced MUC18 expression may be modulated in part through transcription factor specificity protein 1. Overexpression of human MUC18 in HEK293 cells increased cell-associated Mp and NTHi levels. Moreover, MUC18 was shown to directly interact with Mp and NTHi. These results for the first time show that an allergic airway milieu (e.g., IL-13) increases MUC18 expression, which may contribute to increased bacterial infection/colonization in asthma and other lung diseases.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
MUC18 protein immunostaining in human lung tissues and brushed bronchial epithelial cells. (A) Lung tissues from a subject with fatal asthma and a subject without lung disease (normal control) were immunostained using a rabbit anti-human MUC18 antibody (Epitomics, Inc., Burlingame, CA). As compared with the normal airway, fatal asthmatic airway epithelium demonstrated increased MUC18 protein staining, predominantly at the apical surface. No staining was seen in the same fatal asthmatic airway tissue incubated with a rabbit IgG (negative control). Original magnification, ×200. (B) Brushed noncultured bronchial epithelial cells on cytospin slides were immunostained with the same anti-MUC18 antibody as described for the lung tissue. The percentage of MUC18 (+) cells in subjects with mild to moderate asthma (n = 7) was significantly higher than that in normal subjects (n = 4). The horizontal bars represent medians. Original magnification, ×400.
Figure 2.
Figure 2.
Effects of IL-13 on MUC18 expression of cultured bronchial epithelial cells obtained through bronchial brushings in normal subjects (n = 4) and subjects with mild to moderate asthma (n = 7). Brushed bronchial epithelial cells were cultured under the air–liquid interface conditions for 16 days in the presence or absence or IL-13 (10 ng/ml) for MUC18 mRNA and protein measurements using real-time quantitative RT-PCR and Western blotting, respectively. Although IL-13 increased MUC18 mRNA levels in normal subjects and in subjects with asthma (A), it only significantly increased MUC18 protein (B and C) in subjects with asthma. Data are expressed as means ± SEM.
Figure 3.
Figure 3.
IL-13 increases levels of Mycoplasma pneumoniae (Mp) associated with NCI-H292 cells. Cells were treated with or without IL-13 (10 ng/ml) for 48 hours, followed by Mp infection (10 CFU/cell) for 24 (A) or 48 (B) hours. After three washes with PBS, cell-associated Mp levels were quantified. n = 4 replicates.
Figure 4.
Figure 4.
Role of transcription factor specificity protein 1 (Sp1) in IL-13–induced MUC18 expression. NCI-H292 cells were transfected with a control siRNA or Sp1 siRNA for 48 hours, followed by cell culture medium (–) or IL-13 (10 ng/ml) treatment for 48 hours. (A) IL-13 treatment in control siRNA cells significantly increased MUC18 mRNA, which was abrogated by MUC18 siRNA. n = 3 replicates. (B) Western blotting of MUC18 and Sp1 demonstrated inhibition of IL-13–induced MUC18 up-regulation by Sp1 siRNA and reduction of Sp1 protein after Sp1 siRNA. n = 3 replicates. (C) Role of Sp1 in IL-13–induced Mycoplasma pneumoniae (Mp) adherence to NCI-H292 cells. Cells with a 48-hour transfection with a control siRNA or Sp1 siRNA were incubated with IL-13 (10 ng/ml) for 48 hours, followed by Mp infection (10 CFU/cell) for additional 24 hours. After three washes with PBS, cell-associated Mp was quantified. Sp1 siRNA, as compared with the control siRNA, significantly reduced cell-associated Mp levels. n = 4 replicates. Data are expressed as means ± SEM.
Figure 5.
Figure 5.
MUC18 overexpression in HEK293 cells increased levels of cell-associated Mp. (A) MUC18 overexpression in HEK293 cells was confirmed using Western blotting. A total of 60 μg of cell lysates from HEK293 cells with or without MUC18 cDNA transfection or 100 ng of recombinant human MUC18 (rhMUC18) protein (Abnova Corp., Walnut, CA) was run on a 8 to 16% gel and probed with a rabbit anti-MUC18 antibody (Epitomics, Inc., Burlingame, CA). (B) HEK293 cells with or without MUC18 overexpression were incubated with Mp (10 CFU/cell) for 24 or 48 hours. After three washes with PBS, cell-associated Mp levels were quantified. As compared with HEK293 cells without MUC18 overexpression, those with MUC18 overexpression demonstrated an increased level of cell-associated Mp. n = 3 replicates. Data are expressed as means ± SEM. (C) A fluorescent microscopic image showing increased Mp predominantly localized to the surface of MUC18-overexpressing cells. HEK293 cells with or without MUC18 overexpression were incubated with Mp (10 CFU/cell) for 24 hours, pelleted, embedded in glycol methacrylate resin, cut into sections at 2 μm thickness, and stained with a rabbit anti-Mp antibody (Abcam, Cambridge, MA), followed by a FITC-labeled secondary antibody. Original magnification, ×400.
Figure 6.
Figure 6.
MUC18 interactions with Mp. (A) Cell lysates of primary normal human bronchial epithelial cells infected with Mp (10 CFU/cell) under air–liquid interface conditions for 7 days were immunoprecipitated with a rabbit anti-MUC18 antibody (Epitomics, Inc., Burlingame, CA) or a rabbit IgG (IgG control) (Abcam, Cambridge, MA). Coprecipitating Mp proteins were detected using an anti-Mp antibody (Abcam). A protein around 65 kD in Mp was shown to interact with MUC18. (B) Direct binding assay of recombinant human MUC18 protein and Mp. A full-length recombinant human MUC18 protein (1 μg/ml) (Abnova Corp., Walnut, CA) or BSA (1 μg/ml, control for MUC18 protein) was coated on a 96-well plate. After washing the plate, Mp (106 CFU in 50 μl PBS) was added. After three washes, a rabbit anti-Mp antibody (Abcam) was added, followed by incubation with a biotinylated anti-rabbit secondary antibody and avidin-biotin-peroxidase. Tetramethylbenzidine was used as a substrate to develop the plate, and the absorbance at 450 nm was obtained. The increased absorbance at 450 nm indicated a direct binding of MUC18 protein to Mp. n = 3 replicates. Data are expressed as means ± SEM.
Figure 7.
Figure 7.
MUC18 interactions with nontypeable Haemophilus influenzae (NTHi). (A) Direct binding assay of recombinant human MUC18 protein and NTHi was similarly performed as described for Figure 6B. The increased absorbance at 450 nm indicated a direct binding of MUC18 protein to NTHi. n = 4 replicates. Data are expressed as means ± SEM. (B) NTHi association with MUC18-overexpressing HEK293 cells. HEK293 cells with or without MUC18 overexpression were incubated with a control IgG or an anti-human MUC18 antibody ABX-MA1 (10 μg/ml) for 2 hours, followed by infection with NTHi (10 CFU/cell) for 24 hours. After three washes with PBS, cell-associated NTHi was quantified. As compared with HEK293 cells without MUC18 overexpression, MUC18-overexpressing HEK293 cells demonstrated an increased level of cell-associated NTHi, which was blocked by the anti-MUC18 antibody treatment. n = 3 replicates. Data are expressed as means ± SEM.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jean D, Gershenwald JE, Huang S, Luca M, Hudson MJ, Tainsky MA, Bar-Eli M. Loss of AP-2 results in up-regulation of MCAM/MUC18 and an increase in tumor growth and metastasis of human melanoma cells. J Biol Chem 1998;273:16501–16508. - PubMed
    1. Lehmann JM, Riethmuller G, Johnson JP. MUC18, a marker of tumor progression in human melanoma, shows sequence similarity to the neural cell adhesion molecules of the immunoglobulin superfamily. Proc Natl Acad Sci USA 1989;86:9891–9895. - PMC - PubMed
    1. Boneberg EM, Illges H, Legler DF, Furstenberger G. Soluble CD146 is generated by ectodomain shedding of membrane CD146 in a calcium-induced, matrix metalloprotease-dependent process. Microvasc Res 2009;78:325–331. - PubMed
    1. Xie S, Luca M, Huang S, Gutman M, Reich R, Johnson JP, Bar-Eli M. Expression of MCAM/MUC18 by human melanoma cells leads to increased tumor growth and metastasis. Cancer Res 1997;57:2295–2303. - PubMed
    1. Sers C, Kirsch K, Rothbacher U, Riethmuller G, Johnson JP. Genomic organization of the melanoma-associated glycoprotein MUC18: implications for the evolution of the immunoglobulin domains. Proc Natl Acad Sci USA 1993;90:8514–8518. - PMC - PubMed

Publication types

MeSH terms