Gene expression profiling of ovine keratinocytes stimulated with Psoroptes ovis mite antigen-a preliminary study
Authors
Watkins CA, Mackellar A, Frew D, Mackie C, George A, Hopkins J, Burgess ST, McNeilly TN, Huntley JF.
Institution
Moredun Research Institute, Pentland Science Park, Bush Loan, Penicuik
Country
United Kingdom
Year
2009
Journal
Parasite Immunology
Abstract
Sheep scab is caused by the noninvasive mite, Psoroptes ovis, which initiates a profound pro-inflammatory skin response leading to lesion development. To investigate these early events between the skin and the parasite, primary ovine epidermal keratinocyte cultures were generated and challenged with mite derived antigens. The kinetics of the mRNA response of these cells were monitored by microarray. The results indicated that the cells responded within 1 h of challenge, with a significant increase in the pro-inflammatory cytokine IL-8. This result was confirmed by real-time RT-PCR, and showed that IL-8 up-regulation was maximal at 1 h but declined to pre-stimulation levels at 24 and 48 h. The IL-8 mRNA response to mite wash antigens containing secretory and/or excretory proteins was also investigated and compared to the response to whole mite antigen. These studies revealed that the mite wash antigen, at a challenge dose of 10 microg/mL, was markedly more potent and induced significantly higher levels of IL-8 mRNA than the same concentration of whole mite antigen. These results are discussed in relation to mite establishment and survival on the ovine host.
Product use
Isolation and cultivation for staining Involucrin, stimulation with whole mite Antigen, total RNA extraction, RNA amplification, preparation of labeled cDNA and microarray hybridization, Real-time PCR
Tissue type
Epidermal
Tissue info
Skin tissue from the inner thigh of two 6-month-old lambs