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. 2009 May;35(4):272-83.
doi: 10.1080/01902140802635517.

The chemokine CXCL16 is highly and constitutively expressed by human bronchial epithelial cells

Caroline Day  1 , Rashmika Patel, Cristina Guillen, Andrew J Wardlaw
Affiliations

The chemokine CXCL16 is highly and constitutively expressed by human bronchial epithelial cells

Caroline Day et al. Exp Lung Res. 2009 May.

Abstract

The chemokine receptor CXCR6 is highly expressed on lung-derived T cells compared to blood T cells, especially in inflammatory diseases characterised by T-cell migration to the lung. This suggests that CXCR6 is a candidate lung homing receptor. The sole ligand of CXCR6, CXCL16, has previously been shown to be expressed by alveolar macrophages. The authors hypothesized that also structural lung cells express CXCL16. CXCL16 expression was detected using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. Chemotaxis assays were used to test functionality of the secreted protein VSports手机版. Human bronchial epithelial cells secreted relatively high basal levels of CXCL16 (> 1000 pg/mL). Interferon (IFN)-gamma, but not tumor necrosis factor (TNF)-alpha or interleukin (IL)-4, caused a modest but significant up-regulation in secretion. Airway smooth muscle and fibroblasts also expressed CXCL16, but at lower levels. Western blotting detected expression of the full-length (60-kDa) form of the chemokine in cell lysates, and the cleaved (35-kDa) form in culture supernatants. Concentrated supernatants from a bronchial epithelial cell line (BEAS-2B) were chemotactic for CXCR6 expressing T cells from blood. In conclusion, these results suggest that the bronchial epithelium is an important source of constitutively expressed CXCL16, which may be involved in T-cell recruitment to the lung in health and disease. .

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Figures

FIGURE 1
FIGURE 1
(A) Concentrations of CXCL16 in 24-hour culture supernatants from BEAS-2B cells, measured by ELISA. CXCL16 was significantly up-regulated after IFN-γ stimulation (n = 6). (B) Confirmation by PCR of CXCL16 expression on BEAS-2B cells by the presence of a band at 118 bp. Lane 1 corresponds to 100bp DNA ladder, lane 2 corresponds to unstimulated cells, and lane 3 corresponds to IFN-γ–stimulated cells. (C) Real-time PCR amplification plot (left panel) and chart (right panel) showing a 2.2 times up-regulation of CXCL16 mRNA expression when BEAS-2B cells were stimulated with IFN-γ (filled symbols), compared to unstimulated cells (open symbols). GAPDH was used as a normalising housekeeping gene (representative of n = 2). (D) Flow cytometry of BEAS-2B cells detected the membrane-bound form of CXCL16, as demonstrated by a shift in the population when cells had been stained with anti-CXCL16 antibody (shaded peak) as compared to isotype control (blank peak) (representative of n = 5).
FIGURE 2
FIGURE 2
Expression of CXCL16 in primary cultures of human structural lung cells. (A) Levels of CXCL16 in 24h culture supernatants of human bronchial epithelial cells (n = 6), fibroblasts (n = 4), and smooth muscle cells (n = 5) were measured by ELISA in unstimulated cultures (white bars) and after stimulation with 5 ng/mL of IFN-γ (black bars). (B) No significant was observed difference between CXCL16 levels measured in primary human bronchial epithelial 24-hour culture supernatants from nonasthmatic (n = 3) and asthmatic donors (n = 3).
FIGURE 3
FIGURE 3
Western blot showing two forms of CXCL16 are produced by BEAS-2B cells. The full-length form of CXCL16 was found in the cell lysates of the BEAS-2B cells, as demonstrated by a strong band at 60 kDa. The truncated form of CXCL16 was mainly detected in the culture supernatants, as a band at 35 kDa (representative of n = 3).
FIGURE 4
FIGURE 4
Chemotaxis of peripheral blood mononuclear cells stimulated with IL-2 for 7 days, to 10× concentrated BEAS-2B culture supernatants is expressed as the percentage of cells migrating of the cells added to the upper chamber. Chemotaxis was significantly blocked when T cells were preincubated with anti-CXCR6 (but not with the isotype control) (n = 10)), and pertussis toxin (n = 3).
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