Stonehouse, Timothy James; (1998) Analysis of the role of RXR[alpha] in monocyte-macrophage differentiation and function using U937 monoblastoid cells. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

In this thesis the U937 monoblast cell line has been used as a model for investigation of the immunobiology of the mononuclear phagocyte system, both in terms of differentiation, and in terms of functional properties, such as phagocytosis and accessory cell activity. Retinoid X receptor-a (RXRa), a member of the nuclear receptor superfamily, was shown to be expressed in primary human monocytes, and its expression was increased upon differentiation in response to human serum. Expression of RXR dimeric partners, RARa and RAR, was not induced in the same way. Expression of these proteins was also assessed in previously derived novel U937 transfectants that hyper-expressed (aG2S cells), or hypo-expressed (aB5A cells), RXRa. Retinoic acid (RA) activation of aG2S cells led to an altered pattern of expression of surface antigens which suggested a more mature phenotype, when compared to aB5A cells and an empty vector control cell line (MEP). RA-treated aG2S cells were also more efficient at phagocytosing opsonised-SRBC. This immunophagocytic process was inhibited in all three cell lines by prior addition of CD13, CD16 and CD18 mAbs. The increase in efficiency of phagocytosis correlated with a more intense and altered phosphotyrosine response during phagocytosis. Phagocytosis induced activation of members of the MAPK family of kinases in all three cell lines, and phosphoproteins from the U937 transfectants interacted differentially with a panel of SH2 domain fusion proteins. RA-treated aG2S cells were also resistant to ionising radiation-induced cell death. This phenomena was RA-dependent, as vitamin D treatment did not have the same effect. To examine accessory cell function of both the U937 parent cell line and the U937 transfectants, a model U937-T-cell interaction assay was developed, where the cell line provides a co-stimulatory signal for CD3- mediated T-cell activation, independent of CD80/CD86/CD28 interaction. A 2-integrin (CD11a/18)/ICAM-l (CD54) interaction was implicated, and there was evidence to suggest the dominant response was in cells with a CD8+ phenotype. This implied a reduced requirement for CD28- mediated signalling by CD8+ T-cells, and this was confirmed since proliferation of the CD4+ T-cell population increased significantly in response to an added CD28 mAb. CD1la/18 mAbs inhibited U937/T-cell cluster formation as well as proliferation. CD45 mAbs increased the size of the U937/T cell clusters formed, suggesting that ligation of CD45 may be important in the targeting of T-cells towards antigen presenting cells. MAbs to CD53, CD98 and CD147 inhibited U937 dependent T-cell proliferation. CD53 mAbs were not inhibitory when pre-pulsed on to U937 cells, suggesting an effect at the T-cell level. CD98 and CD147 mAbs were inhibitory in pre-pulsing, but also inhibited an accessory cell independent proliferation assay, suggesting these molecules play a role both at the accessory cell level and at the level of the T-cell. The observation that most inhibitory antibodies induce tyrosine phosphorylation in both U937 and T-cells was consistent with this finding. Finally, aG2S cells induced greater T-cell proliferation when used as an accessory cell, than MEP cells, which itself was more efficient than aB5A cells. Previous exposure to opsonised-SRBC had no effect on the accessory cell capabilities of these cells, demonstrating that phagocytic and accessory cell functions are not mutually exclusive.

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