Toll-like receptor agonists induce inflammation and cell death in a model of head and neck squamous cell carcinomas

Abstract

Toll-like receptors (TLRs) are increasingly implicated in the pathogenesis of cancer. The present study describes TLR expression and function in healthy and malignant airway epithelial cells. The squamous cell carcinoma cell line Detroit-562 was compared with the healthy bronchial epithelial cell line NL-20 and primary human nasal epithelial cells (HNECs). TLR2, TLR3 and TLR5 were present in primary head and neck squamous cell carcinomas (HNSCCs). Consistent with this, Detroit-562 expressed TLR2, TLR3 and TLR5, whereas NL-20 expressed mainly TLR3 and HNECs expressed TLR2-5. In Detroit-562, Pam(3)CSK(4), poly(I:C) and flagellin, ligands for TLR2, TLR3 and TLR5, respectively, induced an up-regulation of intercellular adhesion molecule 1 (ICAM-1), an increase in interleukin (IL)-6 and IL-8 secretion and a decrease in cell viability. Additionally, poly(I:C) affected IL-1beta production and the migratory behaviour of Detroit-562. NL-20 responded with a slight increase in IL-8 secretion upon poly(I:C) stimulation. Poly(I:C) induced a small increase in IL-1beta, IL-6 and IL-8 production in HNECs, while Pam(3)CSK(4) increased viability. The TLR signalling was transcription-dependent, but the pathways involved differed among TLRs as well as cells. In Detroit-562, TLR2 and TLR5 activation was mediated via c-jun N-terminal kinase (JNK)-, p38-, phosphatidylinositol 3-kinase (PI3K)- and nuclear factor (NF)-kappaB-related pathways, while TLR3 was dependent on NF-kappaB. In NL-20, TLR3 signalled via p38, and in HNECs, NF-kappaB, JNK and extracellular signal-regulated kinase (ERK) appeared to be involved. We found that TLR agonists induced a robust response in HNSCCs, characterized by generation of inflammation and cell death. A similar response was not seen in normal epithelial cells. Thus, the TLR system should be considered an important target in future antitumour immunotherapy.

Figure 1

Head and neck squamous cell carcinomas (HNSCC) express Toll-like receptor 2 (TLR2), TLR3 and TLR5. The sections show keratinized squamous cell carcinomas from the larynx. The tumour sections were stained with antibodies against (a) TLR2, (b) TLR3 and (c) TLR5. For localization, the sections were stained with 3,3′-diaminobenzidine (DAB; brown staining) and analysed by microscopy (magnification ×4, ×10 and ×40). The arrows indicate TLR-positive cells.

Figure 2

The Toll-like receptor (TLR) expression profiles in Detroit-562 and NL-20. The epithelial cell lines Detroit-562 and NL-20 were analysed for expression of TLR mRNA and proteins using real-time reverse transcription–polymerase chain reaction (RT-PCR) and fluorescence-activated cell sorting (FACS) analysis, respectively. (a, b) TLR mRNA expression in (a) Detroit-562 and (b) NL-20. The values are expressed in relation to the housekeeping gene β-actin as (n = 6). (c, d) Intracellular staining with monoclonal antibodies against TLRs (open histograms) or appropriate isotype controls (shaded histograms) in (c) Detroit-562 and (d) NL-20. Data show one representative out of three independent experiments.

Figure 3

Toll-like receptor (TLR) agonists induce up-regulation of intercellular adhesion molecule 1 (ICAM-1) in Detroit-562. The cells were stimulated with or without Pam₃CSK₄, poly(I:C), flagellin or tumour necrosis factor (TNF)-α (10 ng/ml) for 6 or 24 hr. Thereafter, the cells were stained with monoclonal antibodies against ICAM-1 and analysed by fluorescence-activated cell sorting (FACS). The mean fluorescence intensity (MFI) of the ICAM-1-positive cells was calculated. (a) ICAM-1 expression after stimulation for 24 hr. (b) Time-dependent up-regulation of ICAM-1 by poly(I:C) (C = control; n = 4; *P < 0·05; **P < 0·01; ***P < 0·001).

Figure 4

Toll-like receptor (TLR) ligands induce cytokine secretion. Cells were stimulated with or without Pam₃CSK₄, poly(I:C), flagellin or tumour necrosis factor (TNF)-α (10 ng/ml) for 24 hr. Thereafter, interleukin (IL)-1β, IL-6 and IL-8 production was measured in the cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). (a) IL-1β, (b) IL-6 and (c) IL-8 secretion by Detroit-562 (n = 6). (d) IL-8 secretion by NL-20 after poly(I:C) stimulation (C = control; n = 6; *P < 0·05; **P < 0·01; ***P < 0·001).

Figure 5

Poly(I:C) affects migration of Detroit-562. Cells were plated on a cell migration plate (100 000 cells/well) and incubated in the absence or presence of poly(I:C) (early addition). After 24 hr, the inserts were removed, and the cells were washed and cultured in medium with or without poly(I:C) (late addition) for another 24 hr. Thereafter, cells were stained with Giemsa staining and analysed by microscopy. Results show migration for (a) untreated cells, (b) early addition of poly(I:C), and (c) late addition of poly(I:C). (d) Percentage of migrated area (n = 6; *P < 0·05; **P < 0·01).

Figure 6

Responses induced by Toll-like receptor (TLR) ligands in human nasal epithelial cells (HNECs). (a) HNECs were analysed for expression of TLR mRNA using real-time reverse transcription–polymerase chain reaction (RT-PCR). The values are expressed in relation to the housekeeping gene β-actin (n = 5). (b) The cells were cultured with or without Pam₃CSK₄, poly(I:C), flagellin or tumour necrosis factor (TNF)-α (10 ng/ml) for 24 hr. Thereafter, cells were stained with monoclonal antibodies against intercellular adhesion molecule 1 (ICAM-1) and analysed by fluorescence-activated cell sorting (FACS). The mean fluorescence intensity (MFI) of the ICAM-1-positive cells was calculated (n = 4). (c, d, e) After stimulation, the cell culture supernatants were analysed for (c) interleukin (IL)-1β, (d) IL-6 and (e) IL-8 production by enzyme-linked immunosorbent assay (ELISA) (C = control; n = 6; *P < 0·05; **P < 0·01).

Figure 7

Mitogen-activated protein kinase (MAPK)-dependent Toll-like receptor (TLR) signalling in Detroit-562, NL-20 and human nasal epithelial cells (HNECs). Cells were pretreated with vehicle [dimethyl sulphoxide (DMSO)] or MAPK inhibitors for 1 hr, and further incubated for 24 hr with TLR ligands. The supernatants were then collected and analysed for interleukin (IL)-8 by enzyme-linked immunosorbent assay (ELISA). (a, b, c) Detroit-562 stimulated with (a) 1 μg/ml Pam₃CSK₄, (b) 1 μg/ml flagellin or (c) 10 μg/ml poly(I:C). (d) NL-20 and (e) HNECs stimulated with 10 μg/ml poly(I:C) (n = 4; *P < 0·05; **P < 0·01; ***P < 0·001). ERK, extracellular signal-regulated kinase; JNK, c-jun N-terminal kinase; PI3K, phosphatidylinositol 3-kinase; NF-κB, nuclear factor kappa B.

Figure 8

Nuclear factor (NF)-κB-dependent Toll-like receptor 3 (TLR3) activation in Detroit-562. Cells were stimulated with or without poly(I:C) (10 μg/ml) for 1 hr. Proteins were extracted from the nucleus as well as the cytosol and further analysed for NF-κB by enzyme-linked immunosorbent assay (ELISA). NF-κB was determined in (a) Detroit-562, (b) NL-20 and (c) human nasal epithelial cells (HNECs) (nuclear/total) (n = 3–4; *P < 0·05).