Interleukin-1 Induced Activation of Indoleamine 2,3-Dioxygenase in Human Peripheral Mononuclear Cells
An assay was set up to quantify the activity of indoleamine 2,3-dioxgenase (IDO) in human peripheral mononuclear cells(PMC). Interleukin-1 (IL-1), interferon-gamma (IFN-G) and D-ala peptide T (pep-T) were used to stimulate PMC in culture medium. Following a 44 h incubation period, PMC were pulsed with radiolabeled tryptophan in fresh culture medium. After a period of 4 h, PMC supernatants (SN) were aspirated, microfuged, injected into a C18 column and run through reverse-phase high performance liquid chromatography (HPLC) in an attempt to separate tryptophan and its catabolites. Fractions were collected at 30 s intervals and analyzed by means of scintillation spectrometry. Counts per minute (CPM) were plotted as a function of time for each fraction. Percent 100 specific tryptophan catabolism (%SC) was calculated for each biological response modifier (BRM) induced and control run. These data indicated that, although 2 separate HPLC elution systems were used, a satisfactory separation of radiolabeled tryptophan and its catabolites was not achieved. However, compared to the control run for each PMC donor, some tryptophan degradation may have occured in both IL-1 and IFN-G induced PMC, which would indicate IDO activation. Additional chemical analysis or a different HPLC elution method is needed to confirm whether or not IL-1, IFN-G and pep-T induce IDO activation in PMC.
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