THERE IS NO REGULATORY ROLE FOR INDUCED NITRIC-OXIDE IN THE REGULATION OF THE IN-VITRO PROLIFERATIVE RESPONSE OF BOVINE MONONUCLEAR-CELLS TO MITOGENS, ALLOANTIGENS OR SUPERANTIGENS

Nitric oxide (NO) is a potent cellular mediator which has been shown t o modulate several immune mechanisms. Between species, however, there are considerable differences regarding the signals required for induct ion of NO as well as the kind of cells capable of producing NO. The ob ject of this study was to determine the kinetics of NO production of b ovine blood mononuclear cells (boMNC) stimulated in vitro and to inves tigate whether it modulates their proliferative response following all ogeneic (mixed leukocyte cultures, aMLC), mitogenic (PWM, Con A) or su perantigenic (SEA, SEE) stimulation. NO production was indirectly dete rmined with the Griess reagent measuring nitrite (NO2-). Significant b ut low amounts of NO could be detected as early as day 3 after in vitr o stimulation and did only slightly increase during the 6-8 day cultur e period. Superantigens (SEA, SEE) and aMLCs (4.3-5.2 PM NO2-) induced a significantly higher nitrite accumulation compared to Con A (2.6 PM NO2-). Generation of nitrite, most likely produced by monocytes/macro phages, could be inhibited by 1 mM N-monomethyl-L-arginine (NMLA). Flo w cytometric characterization of various cellular responses revealed n o differences between cultures with or without NMLA. This included the determination of blastogenesis, absolute numbers of viable cells, exp ression density of activation markers (MHC class II, IL-2R alpha) and cellular subpopulations (CD4(+), CD8(+), sIg(+)) among blasts. In addi tion, exogenously provided NO via SNOG in non-toxic concentrations (10 (-5)-10(-4) M) did not alter the proliferative reaction of boMNC in vi tro. The results suggest that NO is induced after in vitro stimulation of boMNC, however, at a low level, and without having any positive or suppressive effects on the so far tested cellular parameters of activ ation and proliferation.