Lipopolysaccharide stimulates the expression of pro-opiomelanocortin mRNA in chicken macrophages, as demonstrated with a competitive polymerase chain reaction
2002
Gerets, H.H.J. | Peeters, K. | Gucht, E. van der | Vandesande, F. (Catholic Univ. of Leuven (Belgium) | Lab | of Neuroendocrinology and immunological Biotechnology) | Berghman, L.R.
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 HD 11 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 per 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 HD 11 POMC mRNA expression showed an increase of one order of magnitude following treatment with 100 ng per ml LPS as compared with untreated controls, no significant differences could be observed after treatment with 50 and 10 ng per ml LPS. Quantitative measurement of POMC mRNA levels is a first step towards a better understanding of the physiological role of non-hypophysial POMC-derived peptides in the response to immune stress in birds.
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