NO involvement in CTX-induced inhibition of T-cell proliferation.

(A) iNOS mRNA analysis by RT-PCR. 0.4 × 10⁶cells/well were cultured for 24 hours in the presence of Con A (1μg/mL) or anti-CD3 (2μg/mL) plus IL-2 (50 units/mL) in the same conditions used for proliferative assays, and iNOS or control G3PDH mRNA was analyzed by RT-PCR as previously. The figure also shows PCR-positive and -negative controls. (B) The effect of NOS inhibitors on T-cell proliferation. Day 6 or 10 after the last dose of CTX, T-cell proliferative activity induced with Con A (1μg/mL) or anti-CD3 (2 μg/mL) plus IL-2 (50 units/mL) was assessed in the absence or the presence of LMMA (0.5 mmol/L), L-NIL (0.5 mmol/L), or AMG (0.5 mmol/L) added at culture initiation. The cells were cultured for 72 hours, and proliferation was estimated by [³H]TdR incorporation in the last 18 hours of culture. Data are the mean ±SEM of triplicate cultures from 3 mice injected i.p. with CTX. At the concentrations used, iNOS inhibitors did not have any significant effect over proliferation of control SC cultures that ranged between 100 × 10³ and 150 × 10³ cpm (not shown). (C) Nitrite accumulation in culture supernatants from SC cultures. Nitrite was measured in the very same cultures used for proliferation assays after 72 hours of culture. As above, data are expressed as the mean ±SEM. Nitrite production by control SC cultures was always <3 μmol/L.