Lipopolysaccharide stimulates the expression of pro-opiomelanocortin mRNA in chicken macrophages, as demonstrated with a competitive polymerase chain reaction
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The goal of the present study was to develop a competitive PCR assay to measure changes in the expression of pro-opiomelanocortin (POMC) mRNA in myelomonocytic HD11 cells after Salmonella typhimurium lipopolysaccharide (LPS) stimulation. Pro-opiomelanocortin mRNA could be detected in HD11 cells by reverse transcription - polymerase chain reaction (RT-PCR). To our knowledge, this is the first observation of a POMC mRNA transcript in avian macrophages. Based on this observation, the effect of stimulation with bacterial LPS on the POMC expression in HD11 cells was investigated by means of a competitive PCR assay. For this purpose, the HD11 cells received a one-hour LPS challenge with LPS concentrations ranging from 10 to 100 ng/ml. A PCR MIMIC (consisting of a heterologous DNA fragment flanked by templates for the gene-specific primers) was used as an internal control in the competitive PCR assay. A ten-fold dilution series of the MIMIC was co-amplified with a constant amount of experimental cDNA. While HD11 POMC mRNA expression showed an increase of one order of magnitude following treatment with 100 ng/ml LPS as compared with untreated controls, no significant differences could be observed after treatment with 50 and 10 ng/ml LPS. Quantitative measurement of POMC mRNA levels is a first step towards a better understanding of the physiological role of non-hypophysical POMC-derived peptides in the response to immune stress in birds.
