The Expression of Stem Cell Inhibitor/Macrophage Inflammatory Protein-1alpha and Macrophage Inflammatory Protein-1beta mRNA in Murine Macrophages

Molecular biology

Reid, Alan James

University:University of Glasgow

关键词: Molecular biology;   

Others  :  http://theses.gla.ac.uk/74756/1/11007675.pdf

来源: University of Glasgow

【 摘 要 】

The proliferation of haemopoietic stem cells in the bone marrow is believed to be controlled by competing interactions between positive and negative regulators. The identification and characterisation of specific negative regulators has been the subject of much investigation and one of these, an activity termed the Manchester Inhibitor, was shown to be effective in inhibiting the proliferation of CFU-S cells. The presence of this activity in murine bone marrow was also demonstrated to correlate inversely with CFU-S proliferative status. Purification of the active component of the Manchester Inhibitor showed it to be functionally and antigenically indistinct from Stem Cell Inhibitor / Macrophage Inflammatory Protein-1alpha (SCI/MIP-1alpha), a member of the MIP-1 family of cytokines. Another, closely related, member of this family, Macrophage Inflammatory Protein-1beta (MIP-1beta), is ineffective as an inhibitor of haemopoietic stem cell proliferation but both SCI/MIP-1beta and MIP-1beta have been shown to be effective in a number of inflammatory assays and MIP-1beta may be a specific antagonist of the action of SCI/MIP-1alpha. Understanding the signals and mechanisms controlling the expression of the genes for both SCI/MIP-1alpha and MIP-1beta is of fundamental importance for gaining a clear picture of the role played by these proteins in the physiology of stem cell proliferation regulation and in the inflammatory response. Previous studies with the Manchester Inhibitor had shown that this activity was a product of bone marrow macrophages and that the ability to detect the activity correlated with stem cell quiescence; however, only limited studies had been carried out on the factors regulating the production of this activity. In this project, an in vitro culture system was set up to study the regulation of the expression of the genes for SCI/MIP-1alpha and MIP-1beta. Macrophages cultured in vitro from normal murine bone marrow (bone marrow-derived macrophages, BMDM) were employed as a surrogate for the SCI/MIP-1alpha and MIP-1beta producer cells in the bone marrow. This cultured population has characteristic proliferative properties and was demonstrated to express mRNA for SCI/MIP-1alpha and MIP-1beta and secrete SCI/MIP-1beta protein. Previous, limited, studies had observed the expression of SCI/MIP-1alpha and MIP-1beta mRNA in macrophage-like or monocyte-like cell lines. This study represents the first study of the expression of these genes in untransformed, primary macrophages. The accumulation of SCI/MIP-1alpha and MIP-1beta mRNA was observed to be greatly increased by treatment of BMDM with bacterial endotoxin and this was investigated in more detail. Both SCI/MIP-1alpha and MIP-1beta mRNAs were observed to be induced in a rapid and transient fashion, which was dependent on the early stimulation of transcription, in a manner suggestive of this being a part of the early phase of the response of BMDM to stimulation by endotoxin. The accumulation of both mRNAs was also demonstrated to be independent of de novo protein synthesis, which places the expression of SCI/MIP-1alpha and MIP-1beta within the "immediate early" group of genes. Furthermore, the glucocorticoid hormone hydrocortisone was seen to be able to downregulate the accumulation of both mRNAs and oppose the induction of accumulation by endotoxin. These characteristics of expression are typical of those observed for cytokine genes. The accumulation of mRNA for SCI/MIP-1alpha and MIP-1beta was induced by treatment of BMDM with conditioned medium from the cell line L929, a potent source of the monocyte/macrophage lineage-specific growth factor CSF-1. This induction was again observed to be rapid and transient. Attempts to demonstrate increased accumulation of either of the MIP-1 mRNAs using recombinant human CSF-1 were unsuccessful, possibly due to the poor activity of the preparations used. The macrophage priming/ activating factor IFNgamma, however, was able to induce the accumulation of both of the MIP-1 mRNAs, again in a transient manner, demonstrating that accumulation of both of these mRNAs could be induced by a cytokine as well as endotoxin. Studies in serum-free medium demonstrated the accumulation of both SCI/MIP-1alpha and MIP-1beta mRNA on refeeding in the absence of donor horse serum. Both SCI/MIP-1alpha and MIP-1beta mRNA was again observed to accumulate with a rapid and transient pattern. Donor horse serum was seen to downregulate, and fetal calf serum to upregulate, levels of both mRNAs. The expression of many genes with the characteristics of expression observed for SCI/MIP-1alpha and MIP-1beta in this study has been postulated to be linked to the induction of cellular proliferation. The proliferative characteristics of the BMDM population employed in this study allowed this question to be addressed.