Membrane and soluble CD14 expression in response to lipopolysaccharides exposure in monocytic cell models
Cardiff Metropolitan University
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Background - CD14 is an important cell surface molecule of monocytic cells. It is involved in cellular activation in binding with bacterial endotoxin lipopolysaccharide (LPS). The receptor for lipopolysaccharide LPS (CD14) exists in a membrane-associated (mCD14) and a soluble form (sCDl4). It is interesting to know how different types of LPS could stimulate and have an effect on CD14 in vitro. Therefore, the hypothesis of this study is to determine both mCD14 and sCDl4 in response to different LPS exposure in 4-hour and overnight study respectively, using different monocytic cell models: non-differentiated THP-1, PMA-differentiated THP-1 and MM6. Methods - Flow cytometry would be used to determine fluorescence (x-mean) and to analyse mCDl4 expression after 4-hour and overnight stimulation. Meanwhile, a commercially available ELISA assay would be used to determine sCDl4 expression at same time scale. Results - Minimal changes in mCDl4 expression were observed during the first 4-hour of LPS stimulation in non-differentiated THP-1 monocytes (< 0.02). sCD14 expression had increased after 4-hour however. After overnight, LPS weakly increased mCD14 expression but had shown further increase in sCDl4, with E.coli RA type RH100 being down-regulated, and might be due to apoptosis or cell contamination. PMA-differentiated macrophages had shown down-regulated expression in mCD14 in both 4-hour (< 0.001) and overnight (< 0.001), caused by cleavage of CD14 induced by PMA during differentiation. sCD14 shown increase at 4-hour but also shown further cleavage of sCD14 after overnight. MM6 had insignificant results at 4-hour, mCDl4 had no response to LPS, but in overnight, it had shown up-regulation in both mCD14 (< 0.001) and sCD14 expression, represented a true response, showing that 4-hour was insufficient for MM6 to respond to LPS. Conclusion - Overall, these findings using different monocytic cell model had shown that LPS do has an impact on CD14 expression, with PMA showing shedding effect on both sCDl4 and mCD14, and MM6 showing a late response in CD14 expression. However, further studies would be advice to thoroughly analyse mRNA expression using alternative methods to establish CD14 expression at genomic level and to determine activities inside the nucleus. In relation to septicaemia and septic shock, it maybe useful in modulating the severity of the pathophysiology.
BSc (Hons) Sports Biomedicine and Nutrition
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