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

Abstract

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.

Fig 1

18S (a), CXCL9 (b), CXCL10 (c) and CXCL11 (d) mRNA expression in the H357 cell line. (ifn) = cells that were treated with IFN-γ for 3, 6, 9, 24, 48 or 72 hours; (con) = control cells that were left untreated over the same time period.

Fig 2

a) The concentration of CXCL9 produced by the H357 cell line stimulated with IFN-γ for 3, 6, 9, 24, 48 and 72 hours. b) The concentration of CXCL10 produced by the H357 cell line without (con) or stimulated with IFN-γ (ifn) for 3, 6, 9, 24, 48 and 72 hours. Significant differences in chemokines production between untreated cells and IFN-γ treated cell line are indicated as * = p<0.05 or ** = p<0.01. The ELISA is the result of triplicate experiments, shown with ±SD.

Fig 3

18S (a), CXCL9 (b), CXCL10 (c) and CXCL11 (d) mRNA expression. mRNA expression in 3 different normal human oral keratinocytes (NHOK), either IFN-γ treated for 48 hours (ifn) or control cells that were left untreated for 48hrs (con).

Fig 4. CXCL9/10 production in NHOK strains.

In all the NHOK strains CXCL10 production was highly significantly increased after IFN-γ stimulation compared to untreated cells (a). The level of CXCL9 production in the stimulated cells was also highly significantly increased in all the cell lines compared to resting cells, however, there was a substantially lower concentration of CXCL9 produced compared to CXCL10 in all the primary cells (b).

Fig 5. FACS profile of gated lymphocytes labelled with anti-CD4 and anti-CD45RA.

migrated to a) 100nM CXCL12 (SDF-1alpha) and b) 1μg/ml CXCL10. c)/d) The normalised % migration of input PBMC to CXCL10 and CXCL12. Basal migration is the migration to cell culture medium alone.

Fig 6

a) The linear range of CXCL9 (a), CXCL10 (b), CXCL11 (c) mRNA expression in a representative OLP sample. The samples were removed from the PCR machine every 2 cycles (from cycle 15–33). CXCL9 expression is located at 351 bp. b) The same process as for CXCL9 was carried out. The molecular weight of CXCL10 expression is at 601 bp.

Fig 7. The semi-quantification of the CXC ELR- chemokines in oral inflammation.

a) 18S, b) CXCL9, d) CXCL10 and f) CXCL11 mRNA expression in 6 samples of oral lichen planus (OLP) (as a representative of 12 different cases) and 6 samples of normal oral mucosa (NOM). c) CXCL9, e) CXCL10 and g) CXCL11 mRNA expression for the same cases with a 2:8 ratio of 18S:competitor (2:8 18S).