INDUCTION OF A MACROPHAGE-LIKE NITRIC-OXIDE SYNTHASE IN CULTURED RAT AORTIC ENDOTHELIAL-CELLS - IL-1 BETA-MEDIATED INDUCTION REGULATED BY TUMOR-NECROSIS-FACTOR-ALPHA AND IFN-GAMMA
C. Suschek et al., INDUCTION OF A MACROPHAGE-LIKE NITRIC-OXIDE SYNTHASE IN CULTURED RAT AORTIC ENDOTHELIAL-CELLS - IL-1 BETA-MEDIATED INDUCTION REGULATED BY TUMOR-NECROSIS-FACTOR-ALPHA AND IFN-GAMMA, The Journal of immunology, 151(6), 1993, pp. 3283-3291
We investigated the effects of murine rTNF-alpha, human rIL-1beta, and
rat rIFN-gamma in various concentrations and/or combinations on induc
ible nitric oxide (NO) production in primary cultures of rat aortic en
dothelial cells. Northern blot analysis of total RNA from induced and
control cultures using the cloned mouse macrophage gene of inducible N
O synthase as probe as well as polymerase chain reaction using a speci
fic primer sequence gave a positive signal for activated cells only. A
RNA approximately 4.4 kb of length similar to the inducible form of N
O synthase in macrophages was labeled. The concentration of nitrite as
a stable reaction product of NO in culture supernatants was determine
d 24 h after incubation with the various cytokines. IL-1beta alone (40
to 1000 U/ml) induced formation of increasing amounts of nitrite with
increasing concentrations of IL-1beta present. Neither TNF-alpha alon
e (10 to 2000 U/ml) nor IFN-gamma alone (25 to 500 U/ml) showed signif
icant effects on nitrite production. Simultaneous incubation with low
concentrations of TNF-alpha (less-than-or-equal-to 100 U/ml) and IL-1b
eta abrogated the induction effect of IL-1beta. Conversely, addition o
f high concentrations of TNF-alpha (greater-than-or-equal-to 500 U/ml)
led to near maximal levels of nitrite formation even at lowest IL-1be
ta concentrations (40 U/ml). In addition, simultaneous incubation of e
ndothelial cells with IFN-gamma plus IL-1beta and/or TNF-alpha led to
near maximal NO production of endothelial cells, even at lowest IFN-ga
mma concentrations (25 U/ml). We hypothesize that the regulating effec
t of TNF-alpha may in vivo help to prevent local inflammatory response
s from spreading to intact sites.