Monophosphoryl lipid A: an effective adjuvant for potentiating mucosal immune responses

guanylate cyclase. In the absence of LPS, SNP, with or without NAC, did not increase TNFα production in human neutrophils. However, LPS-dependent TNFα production was increased by SNP (P <0.05) and this effect was further augmented by the addition of NAC (P <0.02). INF-γ greatly increased LPS-induced TNFα production by human neutrophils (P < 0.01), but SNP plus NAC further increased TNFα production in these highly activated cells (P <0.001). The cell membrane permeable cGMP analog, dibutyryl cGMP (Bt2cGMP) did not increase TNFα production by neutrophils under any of the experimental conditions. These data suggest that NO increases TNFα production through a mechanism independent of cGMP.1 To exclude possible nonspecific effects associated with exogenous NO sources, we transfected human monoblastoid U937 cells with an expression vector containing murine iNOS cDNA.2 Western blot confirmed expression of iNOS protein in these cells, but not in naive or emptyvector transfected cells. Further, iNOS transfectants, but not empty-vector transfectants, spontaneously produced NO (P <0.001). A NOS inhibitor, Nω-methyl-L-arginine (L-NMA), blocked NO release from iNOS transfected cells, as measured by nitrite and nitrate accumulation (P <0.01). After differentiation with phorbol-12-myristate13-acetate (PMA), iNOS transfectants retained the ability to release NO and produced more TNFα (124.9 ± 25.4 pg/5 x 105 cells per 24 h) than did empty-vector transfected cells (21.9 ± 1.9 pg/5 x 105 cells per 24 h; P = 0.02), an effect inhibited by 500 μM L-NMA (54.4 ± 3.1 pg/5 x 105 cells per 24 h; P = 0.05). These results showed that endogenously produced NO, like exogenous NO sources, can upregulate TNFα production in human phagocytes.2 In the next investigation we sought to determine the cGMP-independent mechanism by which NO regulated TNFα production.3 NO has been shown to inhibit adenylate cyclase in cell membrane preparations. Since cAMP is also known to reduce TNFα release, we tested the possibility that NO enhances TNFα production by decreasing intracellular cAMP levels. Like previous experiments, PMA (100 nM) was used to induce U937 cells to produce TNFα. Incubation of these cells with 500 μM SNP or Snitroso-N-acetylpenicillamine (SNAP) increased TNFα production by 7.0and 15.6-fold, respectively (P = 0.002). This response, however, was completely unrelated to cGMP as these cells lack an NO-sensitive guanylate cyclase, and cGMP analogs had no effect on TNFα synthesis. In contrast, SNP and SNAP were both found to decrease basal and agonist-stimulated intracellular cAMP concentrations (P <0.01, for all). Furthermore, H89, an inhibitor of cAMP-dependent protein kinase, mimicked the effect of NO by dose dependently increasing TNFα production in the absence (6.5-fold at 30 μM; P = 0.04), but not in the presence of SNAP (P = 0.8). Conversely, a cell permeable cAMP analog, dibutyryl cAMP, blocked SNAP-induced increases in TNFα production and TNFα mRNA levels (P <0.01, for both). These results indicate that NO-induced upregulation of TNFα production in human phagocytes is dependent on decreases in intracellular cAMP.3 Because cAMP is known to regulate promoter activity through both the cAMP response element (CRE) and Sp1 binding sites, the wild-type 1311-base pair human TNFα promoter was compared to CRE or Sp1 deletion mutants for responses to NO, H89, and dibutyryl cAMP. These experiments demonstrated that the Sp1 site mediated the upregulatory effect of NO and H89 on the TNFα promoter.4 Collectively, this series of investigations suggests the following conclusions: (i) NO upregulates TNFα gene transcription; (ii) this effect of NO is cGMPindependent and associated with decreases in intracellular cAMP; and (iii) the Sp1 binding site in the TNFα promoter functions as a NO-response element.