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. 2015 Mar 7:15:19.
doi: 10.1186/s12890-015-0016-0.

Hypoxia down-regulates expression of secretory leukocyte protease inhibitor in bronchial epithelial cells via TGF-β1

Lisa I Påhlman, Annika Jögi, Magnus Gram, Michiko Mori, Arne Egesten

Hypoxia down-regulates expression of secretory leukocyte protease inhibitor in bronchial epithelial cells via TGF-β1

Lisa I Påhlman et al. BMC Pulm Med. .

Abstract

Background: Secretory leukocyte protease inhibitor (SLPI) is a protein with anti-protease and antimicrobial properties that is constitutively secreted from the airway epithelium. The importance of maintaining a balance between proteases and anti-proteases, and robust innate defence mechanisms in the airways, is exemplified by inflammatory lung conditions such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). Both conditions present with a high protease burden in the airways which leads to tissue destruction. These patients also have an impaired innate immune system in the lungs with bacterial colonization and frequent airway infections. Moreover, both diseases are associated with airway hypoxia due to inflammation and mucus plugs. The aim of the present study was to investigate the role of hypoxia on SLPI production from the airway epithelium.

Methods: Primary human bronchial epithelial cells were grown in sub-immersed cultures or as differentiated epithelium in air liquid interface cultures. Cells were incubated at 21% O2 (normoxia) or 1% O2 (hypoxia), and the release of SLPI was analysed with ELISA. RT-PCR was used to study the expression of SLPI and transforming growth factor β1 (TGF-β1).

Results: Hypoxia decreased the constitutive production of SLPI by bronchial epithelial cells. The multifunctional cytokine TGF-β1, which is known to affect SLPI expression, showed increased expression in hypoxic bronchial epithelial cells. When bronchial epithelial cells were exposed to exogenous TGF-β1 during normoxia, the SLPI production was down-regulated. Addition of TGF-β1-neutralizing antibodies partially restored SLPI production during hypoxia, showing that TGF-β1 is an important regulator of SLPI during hypoxic conditions.

Conclusions: The mechanism described here adds to our knowledge of the pathogenesis of severe pulmonary diseases associated with hypoxia, e.g. COPD and CF. The hypoxic down-regulation of SLPI may help explain the protease/anti-protease imbalance associated with these conditions and vulnerability to airway infections. Furthermore, it provides an interesting target for the treatment and prevention of exacerbation in these patients.

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Figures

Figure 1
Figure 1
Hypoxia reduces SLPI expression in differentiated bronchial epithelial cells. SLPI concentrations in the cell medium (A) and rinsing fluid (B) from air liquid interface cultures incubated at 21% or 1% O2. The figure shows means and SEM for at least 3 independent experiments.
Figure 2
Figure 2
SLPI expression in bronchial epithelial cells is reduced in response to hypoxia. A) SLPI concentrations in cell medium from primary human bronchial epithelial cells grown under normal oxygenation (21% O2) or under hypoxic conditions (1% O2) (n = 5). B) SLPI mRNA levels from bronchial epithelial cells incubated at 21% or 1% O2. The values of fold change were calculated by normalizing to the data from the 21% O2 samples. The figure shows means and SEM of eight independent experiments.
Figure 3
Figure 3
SLPI expression is down regulated by TGF-β1. SLPI concentrations in the cell medium from human bronchial epithelial cells stimulated with TGF-β1 (A), or in the absence or presence of 1 ng/ml TGF-β1 (B). The figure shows means and SEM of at least three independent experiments.
Figure 4
Figure 4
Expression of TGF-β1 and SLPI in response to hypoxia. A) Expression of TGF-β1 mRNA in bronchial epithelial cells cultured in 21% or 1% oxygen. The fold changes were calculated by normalizing to data from the 21% O2 samples (n = 4). B) SLPI concentrations in the cell medium from bronchial epithelial cells incubated at 21% or 1% O2 in the presence of a neutralizing antibody against TGF-β1 (50 μg/ml) or an isotype control antibody (n = 5).
Figure 5
Figure 5
Schematic model of hypoxia-driven SLPI expression. During normal oxygenation, the bronchial epithelium constitutively secretes SLPI, both apically and baso-laterally. When the oxygen concentrations decrease, the epithelium increases its expression of TGF-β1, which in turn down-regulates SLPI production.
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