DOI
https://doi.org/10.25772/D5YW-BG43
Defense Date
1988
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Microbiology & Immunology
First Advisor
T. Mohanakumar
Abstract
Sublines were derived from the U937 human monocytic cell line by limiting dilution techniques. Several of the sublines derived in this manner were found to constituitively express la, unlike most U937 cell lines previously examined. Thus the la+ and la- sublines were examined and characterized phenotypically and functionally. Morphologically the sublines, both la+ and la-, were found to be similar to the originally described U937 parent cell line. They were positive for production of non- specific esterase and expressed the HLA phenotype A(3,X), B(51,18), DR(2,X). The sublines were examined by immunofluorescence techniques with a large number of antibodies specific for cell surface structures and found to express MHC class I, MHC class II the major type being HLA-DR and perhaps HLA-DO in small amounts, Fc receptor, TA-1 structure typical of monocytes and T cells, endothelial antigens and CD4. The sublines and parent U937 cell line were found to express few cell surface antigens typical of mature monocytes/macrophages; however, they expressed myeloid antigens typical of early monocytic or promonocytic lineage. Expression of MHC class II by the sublines was confirmed by immunoprecipitation with monoclonal antibody to a framework determinant of human la and SDS-PAGE autoradiography which gave bands at p29:34 for Ia+ sublines but not for Ia- parent U937 cell lines. Cell surface expression of la was increased to a significant degree by treatment with gamma interferon which peaked at 24-48 hours of treatment.
Functionally the parent U937 cell line and several Ia+ sublines were examined in several assays. The la- and Ia+ U937 cells were found to stimulate the generation of specific CTLs in CML assays to approximately the same degree. The Ia+ sublines were found to stimulate in MLR assay, although variable results were obtained and indicated that the U937 parent and subline cells produced factors which were found to be inherently immunosuppressive. The sublines were able to substitute for monocytes by reconstituting the C03 mediated T cell mitogenic response. The Ia+ sublines and the parent U937 cell line (also weakly Ia+) were found to present tetanus toxoid antigen to nylon-wool purified T cells following an overnight pulse with antigen, and this response was found to be significantly abrogated by addition of antibody specific for CD4 and MHC class II, but not MHC class I. Preliminary characterization of the immunosuppressive factor produced by the U937 cell line and the sublines revealed that it was strongly antiproliferative and affected lymphoid cells somewhat more than non-lymphoid cells; that its probable molecular weight was approximately 90,000, it was not inactivated by treatment with trypsin or chymotrypsin, was not immediately inactivated by freezing although dialysis and long term storage diminished its activity, and was partially inactivated by heat treatment at both 56°C and 80°C. Clear indication of soluble lL-1 production by the U937 parent cell line and the sublines was problematic due to the strong inhibition of proliferative assays by supernatants; however, partial inactivation of inhibitory activity by heat treatment as well as partial removal of the inhibitor fraction by gel filtration indicated that lL-1 or a cytokine with lL-1 activity was constituitively produced by the cells. Membrane lL-1 was detected in very low amounts in unstimulated cells and was significantly increased by treatment with phorbol esters but not other immunomodulators. Preliminary examination of Northern blots of HLA-DR alpha and HLA-DQ alpha mRNA production by the parent U937 cells and the sublines revealed that both HLA-DR alpha and HLA-DQ alpha mRNA were detectable for |a+ sublines E11, G4 and G11, that only trace amounts were detectable for relatively Ia- subline E9 and that surprisingly, HLA-DQ alpha was detectable for the 2-1 parent cell line but no HLA-DR alpha mFiNA was detectable. The results of mRNA analysis following gamma interferon treatment, as well as treatment with LPS and phorbol esters, were variable for the different sublines and indicate that in some sublines gamma interferon treatment appears to augment la specific mRNA and in others the levels are decreased indicating that the expression of MHC class II specific mRNA may be differentially regulated in the different sublines.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
9-14-2017
Comments
Scanned, with permission from the author, from the original print version, which resides in University Archives.