PubMed · 7672940
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M Young. Psoriasis.. https://pubmed.ncbi.nlm.nih.gov/7672940/
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We wish to develop an in vitro test system for pyrogenic substances. A major source of pyrogen activity is endotoxin. Here we describe a highly sensitive endotoxin-monitoring system based on cytokine measurement in human cell lines of myelomonocytoid origin. The following measures were taken to develop an endotoxin monitoring system of high sensitivity. (i) Mono Mac 6 (MM6) and THP-1 cells, which both represent advanced stages of myelomonocytic development, were better suited as endotoxin indicators than the more immature U-937 line. (ii) In order to enhance cell surface expression of CD14, a major lipopolysaccharide (LPS) receptor, cells were pretreated for 2 days with calcitriol. (iii) The use of fetal calf serum (FCS) without detectable endotoxin traces was essential for maintaining a high LPS sensitivity. (iv) Selected subclones of either THP-1 or MM6 were significantly more sensitive LPS indicators than bulk cultures from which the clones originated. (v) Based on stimulation indices, a commercial tumor necrosis factor-alpha (TNF-alpha) immunoassay proved to be a more sensitive LPS indicator than other cytokine assays or the expression of procoagulant activity/tissue factor. Thus we were able to eliminate the disadvantage of previous cell line-based systems (i.e., low sensitivity) without loss of reproducibility which is seen when using fresh blood, monocytes or monocyte-derived macrophages. High endotoxin sensitivity is a prerequisite for a test system specifically indicating pyrogen activity, because it permits the testing of substances at higher dilutions, thereby minimizing nonspecific interference.
ERV3 (HERV-R) is a complete human endogenous retrovirus located on the long arm of chromosome 7. LTR-env-gene-spliced mRNA of 9 and 3.5 Kb is widely expressed in human tissues and cells, but gag-pol mRNA has not been found. Further, the env gp70 gene contains an open reading frame throughout its length and its expression has recently been detected as a full-length protein. The highest expression of ERV3 detected so far is in placenta and the lowest in cytotrophoblasts and choriocarcinoma cell lines. In this report we have studied ERV3 mRNA and protein expression in the human monoblastic cell line U-937 during differentiation into monocytes/macrophages. Differentiation of U-937 cells was induced by 1,25a-dihydroxyvitamin D3 (vitD3), retinoic acid (RA), gamma interferon (IFN-gamma) and phorbol-myristate-acetate (PMA-TPA). The expression of ERV3 env mRNA was found to be differentiation-associated, with high expression detected in the late stages of monocytic development. Using TPA, the expression of ERV3 env was detected as 9- and 3.5-kb transcripts by Northern blotting, as mRNA by in situ hybridization and as a cytoplasmic 65-kDa protein by immunofluorescence and Western blots. Low levels of basal expression were found, with up-regulation of both message and protein at 24 to 48 hr after addition of TPA. Induction with vitD3, IFN-gamma and RA produced higher levels of mRNA at earlier time points. It is concluded that the U-937 cell line represents an excellent model system for further studies to study the relationship between ERV3 expression and cellular differentiation.
We investigated the effect of 1 alpha-25-dihydroxyvitamin D3 [1,25-(OH)2D3] on the expression of the 28-kDa heat shock protein gene (hsp28) and the protein kinase C beta gene (PKC beta) in the human myeloid HL-60 leukemic cell variant HL-525, which is resistant to phorbol ester-induced macrophage differentiation. Northern and western blot analysis showed little or no hsp28 gene expression in the HL-60 cell variant, HL-205, which is susceptible to such differentiation, while a relatively high basal level of hsp28 gene expression was observed in the HL-525 cells. However, both cell lines demonstrated heat shock-induced expression of this gene. During treatment with 50-300 nM 1,25-(OH)2D3, a marked reduction of hsp28 gene expression along with an induction of PKC beta gene expression was observed in HL-525 cells. A gel mobility-shift assay demonstrated that the 1,25-(OH)2D3-induced alteration of hsp28 gene expression was associated with decreased binding activity to the vitamin D3 receptor-vitamin D3 response element (VDR-VDRE), whereas binding to the heat shock transcription factor-heat shock element (HSF-HSE) was not altered. Our results suggest that the dual effect of 1,25-(OH)2D3 on hsp28 and PKC beta gene expression is due to the different sequence composition of the vitamin D response element in the promoter region as well as an accessory factor for each gene or that 1,25-(OH)2D3 increases PKC beta gene expression, which, in turn, negatively regulates the expression of the hsp28 gene or vice versa.