The down-regulation of CD4 by cultured monocytes has been observed by our group and by other investigators. Flow cytometric analysis was performed to elucidate factors influencing this phenomenon. The addition of LPS, GM-CSF, M-CSF or IL-10 to monocyte cultures failed to inhibit the decrease in monocyte CD4 expression following overnight culture. The down-regulation was an adherence-independent phenomenon since it was not prevented by culture in Teflon vials. The type of anticoagulant into which the peripheral blood was collected did not appear to be a factor, The presence or absence of lymphocytes within the cultures was also inconsequential. The continued culture of monocytes in a whole blood environment resulted in decreased CD4 down-regulation. Experiments in which CD4 was tagged with alpha-CD4-PE mAb prior to cell culture revealed that the down-regulation observed was the result of CD4 internalization. In an attempt to further understand monocyte CD4 function, we investigated the role of monocyte CD4 in signal transduction. Stimulation of Thp-1 monocytic cells with antibody to CD4 resulted in a Ca2+ flux, as well as in the time-dependent tyrosine phosphorylation of various proteins having molecular weights of approximately 180, 140, 120, 110, 85, 65, 55, 50 and 35 kDa. We identified the 140 and 85 kDa proteins as PLC-gamma1, and the regulatory subunit of PI3-K, respectively. Using immunoprecipitation/Western immunoblotting, we were unable, however, to show any direct association between CD4 and PLC-gamma1, PI3-K, or other signaling proteins. In an attempt to identify proteins capable of associating with the cytoplasmic tail of CD4, we generated a GST-CD4cyt fusion protein for use in far Western blots and immunoprecipitation experiments. In both types of experiments, the GST-CD4cyt fusion protein routinely associated with 45 and 55 kDa proteins. In the immunoprecipitation experiments, a 35 kDa protein was also observed. The above results suggest that the expression of monocytic CD4 is regulated during the differentiation process. Furthermore, the cytoplasmic tail of monocytic CD4 is associated with various proteins which we postulate function in signal transduction, and which may also play a role in CD4 down-regulation.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/9117 |
| Date | January 2000 |
| Creators | Graziani-Bowering, Gina M. |
| Contributors | Filion, L., |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Detected Language | English |
| Type | Thesis |
| Format | 237 p. |
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