Human lung cancer cells express functionally active Toll-like receptor 9

Abstract

Background: CpG-oligonucleotides (CpG-ODN), which induce signaling through Toll-like receptor 9 (TLR9), are currently under investigation as adjuvants in therapy against infections and cancer. CpG-ODN function as Th-1 adjuvants and are able to activate dendritic cells. In humans TLR9 has been described to be strongly expressed in B-lymphocytes, monocytes, plasmacytoid dendritic cells and at low levels in human respiratory cells. We determined whether a direct interaction of bacterial DNA with the tumor cells themselves is possible and investigated the expression and function of TLR9 in human malignant solid tumors and cell lines. TLR9 expression by malignant tumor cells, would affect treatment approaches using CpG-ODN on the one hand, and, on the other hand, provide additional novel information about the role of tumor cells in tumor-immunology.

Methods: The expression of TLR9 in HOPE-fixed non-small lung cancer, non-malignant tissue and tumor cell lines was assessed using immunohistochemistry, confocal microscopy, in situ hybridization, RT-PCR and DNA-sequencing. Apoptosis and chemokine expression was detected by FACS analysis and the Bio-Plex system.

Results: We found high TLR9 signal intensities in the cytoplasm of tumor cells in the majority of lung cancer specimens as well as in all tested tumor cell lines. In contrast to this non-malignant lung tissues showed only sporadically weak expression. Stimulation of HeLa and A549 cells with CpG-ODN induced secretion of monocyte chemoattractant protein-1 and reduction of spontaneous and tumor necrosis factor-alpha induced apoptosis.

Conclusions: Here we show that TLR9 is expressed in a selection of human lung cancer tissues and various tumor cell lines. The expression of functionally active TLR9 in human malignant tumors might affect treatment approaches using CpG-ODN and shows that malignant cells can be regarded as active players in tumor-immunology.

Figure 1

Immunohistochemistry (IHC) (A-C) for TLR9 detected by a mouse monoclonal antibody. Adenocarcinoma of the lung (A). Squamous cell carcinoma of the lung (B). A549 cells (all 600 ×) (C). In situ hybridization (ISH) targeting mRNA of human TLR9 with a digoxigenin-labeled DNA-probe in a squamous cell carcinoma of the lung (600 ×) (D). Immunohistochemical staining of TLR9-expression-levels in nonmalignant (E) and malignant tissues (F) derived from the same lungs an stained by the use of tissue arrays. Results of RT-PCR targeting TLR9 in cell lines (G). M: molecular-weight marker (MW8, Roche). 1: negative control; 2: A549; 3: NCI-H727; 4: BEAS 2b; 5: Mononuclear cells from a healthy human donor. Confocal laser microscopy of A549 cells transiently transfected with a GFP-TLR9 plasmid: Cytoplasmic expression of TLR9 is observable in all cells, while successful transfection led to overexpression of TLR9 resulting in bright GFP signals completely superimposed by the TLR9 antibody signal (H). Nuclear counterstain was performed with TOTO3.

Figure 4

MCP-1 secretion in response to CpG-ODN-stimulation in the presence or absence of TNF-α by HeLa and A549 cells (A). Data are expressed as the mean ± SD (n = 6). Student's t test was used for statistical analysis. RT-PCR targeting mRNA of MCP-1 in human non-small cell lung cancer tissue stimulated with CpG-ODN for 24 h (B) (M = pBR322-Msp1). Lanes 2 and 3, as well as lanes 4 and 5 respectively show results of tissue samples from the same tumors either in the absence or presence of CpG-ODN.

Figure 2

CpG-ODN-stimulation decreases apoptosis in HeLa and A549 cells. Cells were stained with Annexin-V after CpG-ODN-stimulation in the presence or absence of TNF-α and CHX after 24 h (A). Data are expressed as the mean ± SD (n = 6). Student's t test was used for statistical analysis. Representative histograms are shown from experiments with HeLa cells after CpG-ODN-stimulation in the absence (B) or presence (C) of TNF-α and CHX. Caspase 3 expression in HeLa cells is shown after incubation with TNF-α and CHX (D). In the presence of CpG-ODN the expression is decreased (E). The percentage of positive cells in each sample is indicated.

Figure 3

TLR9 expression after CpG-ODN-stimulation in HeLa cells: There is no difference in TLR9 expression with and without CpG-ODN-stimulation after 24 h (A). CpG-ODN partially inhibit downregulation of TLR9 which is induced by TNF-α and CHX (B). FI = fluorescence intensity.