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X. Huang, S. Lighvani, L.D. Hazlett; Real time PCR analysis of TLR–9, IL–1ß and MIP–2 mRNA expression levels in a murine Langerhans cell line (XS52) after stimulation with bacterial oligonucleotides or heat–killed P. aeruginosa. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4952.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Our purpose was: 1) to determine changes in mRNA expression levels for Toll–like receptor 9 (TLR–9), IL–1ß and MIP–2 in skin–derived Langerhans cells (LC) (XS52) after stimulation with synthetic CpG–oligodeoxynucleotides (CpG–ODN) or heat–killed P. aeruginosa (PA); and 2) to establish parameters for testing corneal LC. Methods: LC were cultured in complete RPMI and 5% fibroblast (NS line) supernatant. Synthetic, unmethylated GpC–ODN (control), CpG–ODN and CpG–inhibitor were designed with a ‘GAGCTT’, ‘GACGTT’, and ‘GGCGGG’ motif, respectively. LC were treated with CpG–ODN or heat–killed PA. Controls were similarly treated with GpC–ODN or with CpG–inhibitor. For real time PCR, total RNA was isolated using TRIzol, concentration determined, and mRNA expression fold differences calculated after normalizing to ß–actin. Results: At 6h, a significant upregulation of mRNA expression for TLR–9 (8 vs. 13 fold), IL–1ß (225 vs. 684 fold) and MIP–2 (237 vs. 825 fold) was detected in LC treated with CpG–ODN vs. heat–killed PA, respectively. Treatment with CpG–inhibitor completely blocked mRNA upregulation of TLR–9, IL–1ß and MIP–2 stimulated by CpG–ODN. In contrast, similar treatment with the CpG–inhibitor only partially blocked the stimulatory effects of heat–killed PA. Conclusion: These results suggest that XS52 skin derived LC from BALB/c mice are capable of responding to synthetic CpG–ODN and upregulate TLR–9 and early response cytokines at the mRNA level. It also suggests that heat–killed PA antigen not only upregulates TLR–9, but probably other Toll receptors as well that in turn may contribute to the upregulation of IL–1ß and MIP–2.
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