Respiratory epithelial cells require Toll-like receptor 4 for induction of human beta-defensin 2 by lipopolysaccharide

Abstract

Background: The respiratory epithelium is a major portal of entry for pathogens and employs innate defense mechanisms to prevent colonization and infection. Induced expression of human beta-defensin 2 (HBD2) represents a direct response by the epithelium to potential infection. Here we provide evidence for the critical role of Toll-like receptor 4 (TLR4) in lipopolysaccharide (LPS)-induced HBD2 expression by human A549 epithelial cells.

Methods: Using RTPCR, fluorescence microscopy, ELISA and luciferase reporter gene assays we quantified interleukin-8, TLR4 and HBD2 expression in unstimulated or agonist-treated A549 and/or HEK293 cells. We also assessed the effect of over expressing wild type and/or mutant TLR4, MyD88 and/or Mal transgenes on LPS-induced HBD2 expression in these cells.

Results: We demonstrate that A549 cells express TLR4 on their surface and respond directly to Pseudomonas LPS with increased HBD2 gene and protein expression. These effects are blocked by a TLR4 neutralizing antibody or functionally inactive TLR4, MyD88 and/or Mal transgenes. We further implicate TLR4 in LPS-induced HBD2 production by demonstrating HBD2 expression in LPS non-responsive HEK293 cells transfected with a TLR4 expression plasmid.

Conclusion: This data defines an additional role for TLR4 in the host defense in the lung.

Figure 1

LPS-induced IL-8 and HBD2 expression in A549 and HEK293 cells. (A) A549 or HEK293 cells (3 × 10⁵/ml) were left untreated (control) or stimulated for 24 h with LPS (10 or 50 μg/ml), IL-1β (100 ng/ml) or TNFα (10 ng/ml). Levels of IL-8 in supernatants were measured by ELISA and values are expressed as ng/ml. Assays were performed in duplicate a minimum of three times. Values are expressed as mean+/- S.E. (n = 3). (B) Total RNA was extracted from A549 or HEK293 cells, reverse transcribed into cDNA and used as a template in PCR reactions using HBD2 gene-specific primers. Products were electrophoresed in 1.5% TBE agarose gels containing 0.5 μg/ml ethidium bromide and visualized under UV. + represents positive control PCR reaction and lanes 1–4 represent untreated cells and cells stimulated for 24 hours with LPS (10 μg/ml), IL-1β (100 ng/ml) or TNFα (10 ng/ml), respectively. Gels are representative of three independent experiments using cultures from different time points.

Figure 2

Characterization of A549 and HEK293 cell lines. (A) Duplicate samples of total RNA was extracted from 1 × 10⁶ HEK293 (HEK) and A549 cells, reverse transcribed into cDNA and used as a template in PCR reactions using TLR4, Mal, CD14, MD2 and GAPDH gene-specific primers. Products were electrophoresed in 1.5% TBE agarose gels containing 0.5 μg/ml ethidium bromide and visualized under UV. (B) Western blot analysis of membrane (mem) and cytosolic (cyt) extracts (10 μg) from A549 and HEK293 cells probed with an anti-TLR4 antibody. Data are representative of three separate experiments. (C) For fluorescence microscopy, A549 cells (2 × 104) were grown in chamber slides, Fc-blocked and labelled with anti-TLR4 (clear) or isotype control antibodies (solid) and fluorophore-conjugated detection antibodies. TLR4 expression was quantified by laser scanning cytometry.

Figure 3

LPS-induced HBD2 expression in A549 cells requires TLR4. A459 cells were incubated with an isotype control or anti-TLR4 neutralizing antibody (Anti-TLR4 mAB 5 μg/ml, 30 min) then, (A) left untreated or stimulated with LPS (10 μg) for 4 or 24 hours. Total RNA was extracted, reverse transcribed into cDNA and used as a template in PCR reactions using HBD2 gene-specific primers. Products were electrophoresed in 1.5% TBE agarose gels containing 0.5 μg/ml ethidium bromide and visualized under UV. Gels are representative of three independent experiments or (B) left untreated or stimulated with LPS (10 μg) for 24 hours, Fc-blocked and labeled with anti-HBD2 (solid) or isotype control antibodies (clear) and fluorophore-conjugated detection antibodies. HBD2 expression was quantified by laser scanning cytometry, as described, and data from three experiments is presented. HBD2 expression is expressed as Mean Channel Fluorescence (MCF) + SEM. (* P < 0.05 vs control, † P < 0.05 vs control + LPS).

Figure 4

ΔTLR4 inhibits LPS-induced HBD2 expression in A549 cells. A549 cells (1.5 × 10⁵) were transfected with pcDNA3 (empty vector) or a ΔTLR4 expression plasmid. 24 h post transfection, cells were left untreated or stimulated with LPS (10 μg/ml) for 24 h. (A) Total RNA was extracted, reverse transcribed into cDNA and used as a template in semi-quantitative PCR reactions using HBD2 and GAPDH gene-specific primers. HBD2 expression was given an arbitrary value of 1 in control cells. Data are expressed as mean +/- S.E. and are obtained from three experiments (* P < 0.05 vs control, † P < 0.05 vs control + LPS). (B) Duplicate experiments were performed in cells cotransfected with a HBD2 promoter-linked luciferase reporter plasmid. Cells were lysed and reporter gene activity was quantified by luminometry. Data are expressed as HBD2 luciferase activity (n = 3). (* P < 0.05 vs control, † P < 0.05 vs control + LPS)

Figure 5

TLR4/MD2 transgene expression confers LPS-responsiveness on HEK293 cells. HEK293 cells (1.5 × 10⁵) were cotransfected with MD-2 and CD4/Toll or full-length TLR4 expression plasmids and a HBD2 promoter-linked luciferase reporter gene. Equal amounts of the corresponding empty vector were transfected into control cells such that the total amount of transfected DNA remained constant. Uniform transfection efficiencies were confirmed using a β-galactosidase reporter plasmid. 24 hours post transfection, cells were left untreated or stimulated with LPS (10 μg/ml) then lysed and reporter gene activity was quantified by luminometry. Data are expressed as HBD2 luciferase activity. Assays were performed in duplicate and are representative of at least three separate experiments. (* P < 0.05, ** P < 0.005, *** P < 0.001 vs control).

Figure 6

ΔMyD88 and ΔMal inhibit LPS-induced HBD2 expression in A549 cells. A549 cells (1.5 × 10⁵) were transfected with pCDNA3.1 or pDC304 (empty vectors), ΔMyD88 or Mal P/H expression plasmids as indicated. 24 h post transfection, cells were stimulated with LPS (10 μg/ml) for 24 h. HBD2 expression was measured by semi-quantitative RTPCR. Expression in LPS-treated cells was ascribed a value of 100 %. Data shown are mean+/- S.E. (n = 3). Regarding amounts of transfected DNA, ΔMyD88 + and ++ represent 100 and 200 ng of dominant negative MyD88 plasmid DNA respectively while Mal + and ++ represent 50 and 100 ng of dominant negative Mal plasmid DNA respectively. (* P < 0.05, ** P < 0.01, *** P < 0.005 vs control + LPS)