PHA-activated PBMC Research Articles

Quantitative analysis of serum IL-6 and its correlation with increased levels of serum IL-2R in HIV-induced diseases.

We have devised a luminescence sandwich ELISA for the quantification of IL-6 in both sera and cell culture supernatants, which had a detection limit of 100 fg/ml of test sample. By using the luminescence sandwich-ELISA, low but measurable levels of IL-6 (9.5 pg/ml on average) were found in the sera from normal individuals. The serum levels of IL-6 were elevated in HIV-seropositive asymptomatic carriers (55.5 pg/ml on average), and the IL-6 levels were correlated with the degree of HIV-induced disease progression (AIDS-related complex 106.8 pg/ml on average and (AIDS 283 pg/ml). IL-6 immunoreactivity in the sera of AIDS patients eluted at a 25,000 m.w. major peak, which was biologically active and heat-stable, and a 500,000 m.w. minor peak in size-exclusion HPLC. Interestingly, a significant correlation was observed between the serum IL-6 levels and soluble IL-2R levels. In vitro, HIV infection of PHA-activated PBMC led to enhanced release of IL-6 into the culture supernatants. Moreover, soluble IL-2R release was markedly increased by adding exogenous IL-6, whereas it was decreased by adding the neutralizing anti-IL-6 mAb to the cultures. These results demonstrate that increased IL-6 levels are significantly associated with sIL-2R levels, and suggest a cause of the increased levels of this receptor in patients with HIV infection. Furthermore, both serum IL-6 and serum IL-2R levels in HIV infection reflect the stage of the HIV-induced disease.

Polyamine oxidation down-regulates IL-2 production by human peripheral blood mononuclear cells.

Polyamine biosynthesis by mononuclear cells (MNC) appears to regulate T cell activity since 1) inhibition of polyamine biosynthesis increases IL-2 production and 2) H2O2 (a product of polyamine oxidase, PAO), suppresses lymphocyte proliferation. We investigated this immunoregulatory mechanism and find that catalase, an H2O2 inhibitor, also enhances IL-2 production by MNC. The addition of PAO, but only in the presence of either endogenous or exogenous spermidine (a polyamine), decreases IL-2 production; this effect is catalase inhibitable. Pre-incubation with spermidine, but not any of three diamines tested, suppresses PHA-stimulated IL-2 production and this effect requires monocytes. Three PAO inhibitors have an enhancing effect on IL-2 production which is again monocyte dependent. These results suggest that: 1) H2O2 produced by PHA-activated PBMC down-regulates IL-2 production; 2) products of the interaction between PAO and spermidine inhibit IL-2 production and H2O2 is essential but apparently not sufficient to mediate this effect; 3) human PBMC contain PAO activity. This polyamine-PAO-dependent inhibitory mechanism might constitute a feedback loop that limits human T cell proliferation and, consequently, also the immune responses mediated by these cells.