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. 2011;6(7):e21827.
doi: 10.1371/journal.pone.0021827. Epub 2011 Jul 15.

Innate immune responses to bacterial ligands in the peripheral human lung--role of alveolar epithelial TLR expression and signalling

Andrew J Thorley  1 , Davide Grandolfo, Eric Lim, Peter Goldstraw, Alan Young, Teresa D Tetley
Affiliations

Innate immune responses to bacterial ligands in the peripheral human lung--role of alveolar epithelial TLR expression and signalling

Andrew J Thorley et al. PLoS One. 2011.

Abstract

It is widely believed that the alveolar epithelium is unresponsive to LPS, in the absence of serum, due to low expression of TLR4 and CD14. Furthermore, the responsiveness of the epithelium to TLR-2 ligands is also poorly understood. We hypothesised that human alveolar type I (ATI) and type II (ATII) epithelial cells were responsive to TLR2 and TLR4 ligands (MALP-2 and LPS respectively), expressed the necessary TLRs and co-receptors (CD14 and MD2) and released distinct profiles of cytokines via differential activation of MAP kinases. Primary ATII cells and alveolar macrophages and an immortalised ATI cell line (TT1) elicited CD14 and MD2-dependent responses to LPS which did not require the addition of exogenous soluble CD14. TT1 and primary ATII cells expressed CD14 whereas A549 cells did not, as confirmed by flow cytometry. Following LPS and MALP-2 exposure, macrophages and ATII cells released significant amounts of TNFα, IL-8 and MCP-1 whereas TT1 cells only released IL-8 and MCP-1. P38, ERK and JNK were involved in MALP-2 and LPS-induced cytokine release from all three cell types. However, ERK and JNK were significantly more important than p38 in cytokine release from macrophages whereas all three were similarly involved in LPS-induced mediator release from TT1 cells. In ATII cells, JNK was significantly more important than p38 and ERK in LPS-induced MCP-1 release. MALP-2 and LPS exposure stimulated TLR4 protein expression in all three cell types; significantly more so in ATII cells than macrophages and TT1 cells. In conclusion, this is the first study describing the expression of CD14 on, and TLR2 and 4 signalling in, primary human ATII cells and ATI cells; suggesting that differential activation of MAP kinases, cytokine secretion and TLR4 expression by the alveolar epithelium and macrophages is important in orchestrating a co-ordinated response to inhaled pathogens.

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Conflict of interest statement

Competing Interests: The authors have read the journal's policy and have the following conflicts: Dr. Alan Young is an employee of AstraZeneca, who funded this study. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Effect of LPS and serum on cytokine release from alveolar macrophages, ATII and TT1 cells.
Cells were exposed to LPS (1–100 ng/ml) in the presence and absence of serum for 24 hours. Conditioned media was then aspirated and TNFα, MCP-1 and IL-8 release measured by Luminex. Data expressed as mean ± SE (n = 6 subjects). **P<0.001, ***P<0.0002.
Figure 2
Figure 2. Effect of MALP-2 and serum on cytokine release from alveolar macrophages, ATII and TT1 cells.
Cells were exposed to MALP-2 (1–100 ng/ml) in the presence and absence of serum for 24 hours. Conditioned media was then aspirated and TNFα, MCP-1 and IL-8 release measured by Luminex. Data expressed as mean ± SE (n = 6 subjects). **P<0.002, ***P<0.0001.
Figure 3
Figure 3. FACS analysis of cell surface CD14 expression on primary human ATII cells, immortalised TT1 cells and A549 cells.
Cells were immunostained with FITC-labelled anti CD-14 antibody and expression measured using flow cytometry. Representative hisotgrams demonstrate that ATII cells (A) and TT1 cells (B) express CD14 whereas A549 cells (C) do not.
Figure 4
Figure 4. Comparative expression of CD14 on primary human ATII cells, immortalised TT1 cells and A549 cells.
Cells were immunostained with CD14 and cells surface expression measured using flow cytometry. The ratio of median fluorescent intensity of CD14 staining relative to IgG control was calculated for all cells. Results demonstrated that both ATII cells and TT1 cells express CD14 whereas A549 cells do not. Furthermore, ATII cells had significantly higher CD14 staining than TT1 cells. Data expressed as mean ± SE (n = 4 subjects). **P<0.007.
Figure 5
Figure 5. Effect of CD-14 and MD2 neutralisation on LPS-induced cytokine release from alveolar macrophages, ATII and TT1 cells.
Cells were pre-incubated with an anti-CD14 or anti-MD2 neutralising antibody for 1 hr before exposure to 100 ng/ml LPS for 24 hours. TNFα, MCP-1 and IL-8 release was measured by Luminex. Data expressed as mean ± SE (n = 4 subjects). **P<0.002, ***P<0.0001. The relevant IgG isotype was used as a control and had no effect on LPS-induced mediator release.
Figure 6
Figure 6. Effect of CD-14 and MD2 neutralisation on MALP-2-induced cytokine release from alveolar macrophages, ATII and TT1 cells.
Cells were pre-incubated with an anti-CD14 or anti-MD2 neutralising antibody for 1 hr before exposure to 100 ng/ml MALP-2 for 24 hours. TNFα, MCP-1 and IL-8 release was measured by Luminex. Data expressed as mean ± SE (n = 4 subjects). The relevant IgG isotype was used as a control and had no effect on LPS-induced mediator release.
Figure 7
Figure 7. Inhibition of LPS-induced cytokine release from alveolar macrophages ATII and TT1 cells by MAP kinase inhibitors.
Cells were pre-incubated for 30 min with 10 µM of inhibitors of p38 (SB202190), JNK (SP600125) or ERK (PD98059) before exposure to 100 ng/ml LPS for 24 hours. TNFα (A), MCP-1 (B) and IL-8 (C) release were measured by Luminex. Data expressed as mean ± SE (n = 4 subjects). *P<0.05, **P<0.002, ***P<0.0002.
Figure 8
Figure 8. Inhibition of MALP-2-induced cytokine release from alveolar macrophages ATII and TT1 cells by MAP kinase inhibitors.
Cells were pre-incubated for 30 min with 10 µM of inhibitors of p38 (SB202190), JNK (SP600125) or ERK (PD98059) before exposure to 100 ng/ml MALP-2 for 24 hours. TNFα (A), MCP-1 (B) and IL-8 (C) release were measured by Luminex. Data expressed as mean ± SE (n = 4 subjects). *P<0.05, **P<0.005, ***P<0.0001.
Figure 9
Figure 9. Effect of LPS on alveolar macrophage and ATII cell TLR-4 protein expression.
Alveolar macrophages (A) and ATII cells (B) were exposed to LPS (100 ng/ml) over a three hour time course. Cells were fixed in methanol at 0, 15 mins, 30 mins, 1 hr, 2 hrs and 3 hrs and stained with FITC-labelled anti-TLR4 antibody. TLR4 expression was visualised using confocal microscopy. Images were taken through the centre of the cell to visualise cytosolic as well as cell surface expression. Data representative of 3 separate subjects.
Figure 10
Figure 10. Effect of MALP-2 on alveolar macrophage and ATII cell TLR-4 protein expression.
Alveolar macrophages (A) and ATII cells (B) were exposed to MALP-2 (100 ng/ml) over a three hour time course. Cells were fixed in methanol at 0, 15 mins, 30 mins, 1 hr, 2 hrs and 3 hrs and stained with FITC-labelled anti-TLR4 antibody. TLR4 expression was visualised using confocal microscopy. Images were taken through the centre of the cell to visualise cytosolic as well as cell surface expression. Data representative of 3 separate subjects.
Figure 11
Figure 11. Effect of LPS and MALP-2 exposure on TLR4 expression by alveolar macrophages, ATII and TT1 cells.
Using Simple PCI software analysis of images captured by confocal microscopy, the mean fluorescent intensity of staining of individual cells for TLR4 was measured over a 3 hour time course of exposure to LPS (A) or MALP-2 (B). Data expressed as mean ± SE (n = 3 subjects). *P<0.05, **P<0.002, ***P<0.0001.
Figure 12
Figure 12. Cellular localisation of TLR4 protein in primary human alveolar macrophages and ATII cells.
Under high magnification it can be seen that, in alveolar macrophages (A), there is distinct expression of TLR4 protein at the cell surface at 30 minutes with some intracellular staining. By three hours the intracellular staining has increased greatly (in particular in the perinuclear region). In contrast, in ATII cells there is strong intracellular TLR4 expression at 30 minutes which increases further by 3 hours. Similarly to the alveolar macrophages, intracellular staining is most intense in the perinuclear region. TLR4 expression could not be visualised in ATI cells due to low expression.
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References

    1. Albiger B, Dahlberg S, Henriques-Normark B, Normark S. Role of the innate immune system in host defence against bacterial infections: focus on the Toll-like receptors. J Intern Med. 2007;261:511–528. - PubMed
    1. West AP, Koblansky AA, Ghosh S. Recognition and Signaling by Toll-Like Receptors. Annual Review of Cell and Developmental Biology. 2006;22:409–437. - PubMed
    1. Wyllie DH, Kiss-Toth E, Visintin A, Smith SC, Boussouf S, et al. Evidence for an Accessory Protein Function for Toll-Like Receptor 1 in Anti-Bacterial Responses. J Immunol. 2000;165:7125–7132. - PubMed
    1. Takeuchi O, Kawai T, Muhlradt PF, Morr M, Radolf JD, et al. Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int Immunol. 2001;13:933–940. - PubMed
    1. Medzhitov R, Preston-Hurlburt P, Janeway CA., Jr A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature. 1997;388:394–397. - PubMed

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