Tissue-specific regulation of CXCL9/10/11 chemokines in keratinocytes: Implications for oral inflammatory disease

The IFN-γ-inducible chemokines CXCL9, CXCL10, and CXCL11 play a key role in many inflammatory conditions, particularly those mediated by T cells. Therefore, the production of these chemokines in peripheral tissues could be instrumental in the pathophysiology of tissue-specific immunological diseases such as oral lichen planus (OLP). In the present study, we assessed the production of keratinocyte-derived CXCL9/10/11 under basal and inflammatory conditions and investigated whether these chemokines were involved in the pathogenesis of OLP. We used semi-quantitative PCR, ELISA, chemotaxis assays, and fluorescence-activated cell sorting (FACS) to assess the expression and functional role of CXCL9/10/11 in oral keratinocytes (three strains of normal human oral keratinocytes (NHOK), and the H357 oral cancer cell line) in the presence or absence of IFN-γ. CXCL9/10/11 were also assessed in tissues from normal patients and those with oral lichen planus (OLP). The time course study in oral keratinocytes treated with IFN-γ showed that expression of CXCL9/10/11 chemokines was significantly enhanced by IFN-γ in a time-dependent manner. In particular, CXCL10, a prominent chemokine that was overexpressed by IFN-γ-stimulated NHOK, was able to effectively recruit CD4 lymphocytes, mainly CD4+CD45RA- cells. Significantly higher levels of CXCL9/10/11 were found in tissues from patients with OLP compared to normal oral mucosa. Taken together, the results demonstrate that normal oral keratinocytes produce chemotactic molecules that mediate T cell recruitment. This study furthers understanding of chemokine production in oral keratinocytes and their role in the pathophysiology of oral mucosa, with particular relevance to OLP.

The oral squamous cell carcinoma cell line, H357, was established by Prime et al [41], from a primary explant of a tongue squamous cell carcinoma. This cell line was grown in the same medium as described for the NHOK.

IFN-γ cell treatment assay
In a modification of the method utilised by Altenburg et al [42], the NHOK1, NHOK2, NHOK3 and the H357 cell line (at 2nd or 3rd passage) were seeded at 8x104cells/ well in a Falcon 6 well plate (Becton Dickinson, Oxford, UK) with 3mls of KBM-2 medium containing no hydrocortisone. The cells were incubated for at least 3-5 days until cell culture was 60-80% confluent. We set up the optimal experimental conditions in preliminary experiments with dose-response curves.
Medium containing human recombinant 1000U/ml IFN-γ (catalogue number I3265, purity ≥ 98%, Sigma-Aldrich, Poole, UK) was added to 3 wells and control cell culture medium only was added to the remaining 3 wells. The 1000U/ml concentration of IFN-γ had been successfully used by previous studies to stimulate keratinocytes in vitro [42][43][44][45]. The cells were incubated for 48hrs or, in the case of the H357 time course, for the following time-points: 3hrs, 6hrs, 9hrs, 24hrs, 48hrs and 72hrs. The supernatant was extracted, centrifuged and stored at -70oC. The adherent cells were washed with PBS (Gibco Life Technologies, Paisley, UK) before 0.5ml of Trireagent were added. The suspension was then removed and stored at -70oC. The RNA was isolated as described below. mRNA isolation and semi-quantitative RT-PCR OLP and normal oral mucosa (NOM) tissue were obtained and prepared for RNA isolation. RNA isolation and cDNA synthesis of NHOK, H357, NOM and OLP tissue was carried out. The RNA was extracted according to the manufacturer's instructions, utilising 2ul Pellet Paint Co-precipitant (Novagen, Nottingham, UK) to visualise the RNA pellet. The purified RNA was dissolved into 25ul DEPC water (Ambion, Austin, US) and stored at -70°C.
For each primer the linear range was determined by repeating the above reaction with optimised magnesium concentration for each primer and stopping the reaction at 15,17,19,21,23,25,27,29,31,33 and 35 cycles. The mid-point of each linear range was determined by using intensity analysis of the bands with AlphaImager software, and this cycle length was utilised for each primer in subsequent reactions. 18S primer and 18S Competitor primers (Ambion, Texas, USA) were combined to ratios 1:9, 2:8 and 3:7 respectively. For each of the primers (CXCL9/10/11) 4µl of the 18S primer competitor mix was added to the RT-PCR reaction and the results were compared to the reactions containing the specific primers without any 18S primer. The reaction that had the same level of specific primer expression as that without 18S primer added was selected for quantification. The band intensity of the 18S and of the specific primer was quantified in each sample with Phoretix 1D software (Phoretix, Newcastle, UK).

Enzyme-linked immunosorbant assay (ELISA)
A 96 well maxisorp-surface immunoplate (Nunc, Denmark) was coated overnight with a monoclonal antibody against the human protein to be studied. The plate was then washed 3 times with wash buffer (2.5mMNa2HPO4 (BDH), 0.5mM NaCl (BDH), 7.5mM NaH2PO4.2H2O (BDH) and 0.1% of Tween 20 (BDH). 100ml of cell supernatant or positive control (in a range of dilutions to obtain a standard curve) was added and incubated for 2 hours at room temperature then washed. A biotinylated antibody was used as a secondary antibody; 100µl of this antibody, diluted to an appropriate concentration, was added to each well. The plate was sealed and incubated for 1 hour at room temperature, then washed 3 times. Bound secondary antibody was detected by adding 100µl avidin-HRP (Dako, Denmark) [diluted 1:4000] and incubating for 30 minutes at room temperature. 25ml H2O2 was added to OPD (1 tablet of o-phenyl diaminazadine (Sigma, Poole, Dorset) in 25ml of 34.7mM citric acid, 66.7mM Na2HPO4) and 100µl of this solution was dispensed to each well immediately and incubated at room temperature for 15 mins. The reaction was stopped by adding 100ml of 1M sulphuric acid to the wells and the absorbance measured at 490nM. Chemokine concentration in the supernatant was then extrapolated from the standard curve generated from standards using Revelation software (Dynex Technologies, Virginia, US) attached to an ELISA plate reader (Dynex Technologies, Virginia, US).

Chemotaxis assay
Peripheral blood mononuclear cells (PBMC) were prepared from fresh blood obtained from healthy patients. The lymphocyte separation was carried using Ficoll-Paque