Research Paper Mediators of Inflammation, 62-68 (1996)

TIiE production of nitric oxide (NO) was measured in cultures of spleen cells stimulated by lipopolysaccharide (LPS), IL-2 or LPS + IL-2. We observed that NO synthesis is increased by IFN-1( but inhibited by IFN-/. This is not the case when IL-2 is present in the cultures, since interferons play a minor role in the regulation of the NO production. When IL-2 and LPS were associated in the cultures, the IFN-/ role seems more important than that of IFN-/. PGE2 inhibits NO production in LPS supplemented cultures but has a slight effect in the presence of IL-2 and no effect with IL-2 + LPS. 3-isoButyl-l-methylxanthine OBMX), an inhibitor of phosphodiesterases, induces a decrease of IFN production. In the presence of H-7, an inhibitor of protein kinase C (PKC), NO production is reduced when the cultures are supplemented by LPS or IL-2 but not when IL-2 and LPS are both added. H-7 also reduced IFN production. In the presence of Na-monomethyl-L-arginine (N-MMA), an inhibitor of NO synthesis, IFN production was increased, with no change in the cytotoxic activity. Hence, interferons regulate NO production by mouse spleen cells and, in return, NO modulates the generation of IFN.


Introduction
Nitric oxide (NO) is important in many biological functions. It is generated from L-arginine by the enzyme NO synthase (NOS). Peritoneal macrophages of mice are the best-characterized source of NO.
LPS and interferonq, (IFNq,) constitute the major stimulating factors of NO production 2 by increasing cellular concentrations of NOS. Many functions of LPS have been reported to be mediated by prostaglandin production, which in turn acts by increasing cAMP. 4'5 The role of PGE2 on NO production is unclear. PGE2 did not affect NO production nor did it inhibit NO production. Other work shows that NO activates cyclooxygenase enzymes and leads to a marked increase in PGE2 production. 8 IntraceLlular mechanisms induced by LPS implicate protein kinase C (PKC) and cAMP dependent protein kinase A. LPS causes the translocation of protein kinase C from cytosol to the membrane. 9 PKC has been involved in the induction of NO synthase activity in macrophages by IFNq,. 1 Another report suggested rather the role of protein kinase A.
The studies on NO production and NO regulation have been performed essentially with purl-62 Mediators of Inflammation Vol 5 1996 fled macrophage populations. We have shown in previous work that LAK activity induced by IL-2 was reduced by LPS and that the decrease was related to the increase of IFN-cz/ [3 production. 2 On the other hand, NO was implicated in LAK activity in rodents. Thus the aim of this study was to evaluate NO production induced by splenic macrophage cultures with other splenic cell populations and their cytokines after stimulation by LPS and IL-2, and to assess if there is a relationship between the generation of cytotoxic activity and NO production. We (PBS). Erythrocytes were removed by osmotic shock and the final cell were added. After 18h at 37C, the cells were suspension was resuspended in RPMI 1640 infected with VSV at high multiplicity of infection. culture medium supplemented with 5% foetal The plates were scored after 24h. The antiviral calf serum, t-glutamine (2 mM), Hepes (10 mM), effect was estimated by measuring the incorporapenicillin (100 U/ml), streptomycin sulfate tion of red neutral dye into living cells according (100btg/ml), and 2-mercaptoethanol (0.05mM), to the method described by Hudson and Hay 5 which constitutes complete medium, and using a Titertek multiscan spectrophotometer. The IFN titre of a supernatant was estimated as the reciprocal of the dilution inhi-Measurement of NO production: NO was estimated by measuring the formation of NO-biting 50% of the cytopathic effect.
(the stable oxidative end product of NO) which serves as a quantitative index of macrophage u* activation. NOlevels in culture fluids were estimated by using the Griess reagent. 4 Briefly, Release of NO in spleen cells cultured in the pre-1001.tl culture fluid was incubated with an equal sence ofLPS, IL-2 or LPS + IL-2: Initially we tried volume of 1% sulfanilamide and 1% N-l-naphthy-to determine whether under our experimental lethylenediamine dihydrochloride in 2.5% H3PO4 conditions, splenocytes could be stimulated by (Sigma) at room temperature for 5 min. NO-IL-2, LPS or both, to produce NO. For that was quantified by using spectrophotometry to purpose spleen cells were cultured for 3 days in the presence of increasing concentrations of the measure the OD at 570nm, with NaNO2 as a standard, inducers used either alone or associated. NO- Cytotoxicity as_say: Effector spleen cells were assayed using 51Cr-Na2CrO4-1abelled (Amersham, UK) target cells. Cells from the YAC line were used as targets. Cytotoxic activity was measured concentrations of reagent (lng/ml), increased and reached a plateau for a range of concentrations from 100 to 10000ng/ml. NO induction was possible with 100 U/ml of IL-2, and was even greater when LPS and IL-2 were present in the culture. One should underline that even if IL-2 alone at 10 U/ml did not induce NO synthesis it for different spleen/target cell ratios in a final can act synergistically with LPS. volume of 2001al. After 4h incubation at 37C, In a previous report 12  (1 l.tg/ml). As the aim of this work was to investigate whether NO production was involved in that decrease, the following experiments were performed with 1000U/ml of IL-2 and 1 l.tg/ml of LPS.  12 We have studied the effects of IFNq, or IFN-a/ on NO production induced by LPS after 72 h of culture. For this purpose 100 U/ml of IFNq, or IFN-a/ were added to spleen cells cultured in the presence of LPS at 1 l.tg/ml. Table 1 summarizes four experiments. It was observed that IFNq, induced an increase of NO-(+ 128%) while IFN-t/13 reduced NOproduction (--34%).
We then determined if the interferons induced by LPS, IL-2 or a mixture of both played a role in NO production. For that, the culture was incubated in the presence of anti-IFN-2 antibodies or anti-IFN-a/13 anti-serum, and NOwas measured after 3 days. Table 2 presents the results of three The role of cAMP on NO production by spleen cells cultured in the presence of IL-2 or IL-2 + LPS, in relation to IFN production: LPS-stimulated macrophages produce PGE2 which induces a transient increase of cAMP production. cAMP is degraded by phosphodiesterases. For testing the effects of endogenous PGE2 stimulated by LPS, we added IBMX, an inhibitor of phosphodiesterase, to the cultures and therefore promoted the accumulation of cAMP in the culture medium. NO production was measured after 3 days. Results were presented as the percent variation of NO between the IBMX group and the untreated group. The percent variation of five experiments is shown in Table 3. The produc- NO, absence of implication in cytotoxic activity induced by LPS  tion of NO was inhibited when IBMX was present in LPS-stimulated cultures (--75%), and much less for IL-2 or IL-2 + LPS stimulated cultures, -20% and -16%, respectively. LPS seems to induce cAMP accumulation since the presence of IBMX corresponded to NO inhibition, but it seems that it was not secondary to PGE2 induction. Indeed, when the cultures were incubated in the presence of indomethacin, NO production was reduced similarly to that in cells cultured without indomethacin (data not shown). The addition of IBMX produced a fall of IFN production. The IFN level (which was, respectively, 29

Role of NO in the generation of IAK cells and
IFN production: We have found NO production by spleen cells cultured with LPS, IL-2 or IL-2 + LPS. We have examined if NO is involved in the generation of cytotoxic cells of the LAK type and in IFN production. In these experiments NO, which is derived from a terminal guanidino nitrogen from a_r{.inine, is blocked by the -arginine analogue N-monomethyl arginine (N-MMA).
NO and IFN levels were measured in the supernatants and the cells were collected and tested for their cytotoxic activity against YAC cells for different effector-target cell ratios (10:1 to 0.15:1). Cytotoxic activity was expressed in lytic units. Results of four experiments are reported in Table 5.
We observed that the production of NO falls in the presence of N-MMA, while IFN production was always increased. There was a non-significant trend for cytotoxic activity to increase in the presence of N-MMA.

Discussion
We have examined NO production in murine spleen cell cultures stimulated with either LPS, IL-D. Vaillier, Daculsi and N. Gualde Thus, IFN-a/]3 has immunomodulatory properties which differ from those of IFN-7 for the modulation of NO production. Different effects of these two types of IFN have been described 21 for induction of IL-1; induction of cytotoxic activity 2 and ConA and IL-2-induced proliferation of spleen T-cells. 22 If IFN-, is an inducer of NO, it must be emphasized that NO is an inhibitor of IFN production. Indeed when the cultures were made in presence of an inhibitor of NO, IFN production was increased.
We observe that independent of the stimulating agent, the reduction of NO by N-MMA was followed by a small increase in cytotoxic activity in nine of ten cytotoxic assays (Table 5). In contrast with our results, Juretic et a1.13 reported that reduction of NO production induced a decrease of LAK activity in rodent spleen cells. At present, we do not have an explanation for this discrepancy.
The increase of IFN production and the weak effect on cytotoxic activity in cultures made in the presence of an inhibitor of NO may be secondar to a reduction of PGE2. Indeed Salvemini et al. have shown that PGE2 production was associated with NO production which activates cyclooxygenase enzymes. So we hypothesize that the fall of NO induced a reduction of PGE2, which explains the increase of IFN. PGE2 is known to be an inhibitor of IFN production 23 and cytotoxic activity generation. 24 In our system LPS induced PGE2 because (a) IFN production and cytotoxic activity were increased in the presence of indomethacin, which is an inhibitor of cyclooxygenase pathway; 12 and (b) IBMX which produces a cAMP accumulation inhibits NO production (Table 4). On the other hand exogenous PGE2 inhibits NO induced by LPS (Fig. 2). Unexpectedly, the presence of indomethacin which should provoke an increase of NO synthesis by inhibition of PGE2 production, caused a slight decrease of NO production. A similar effect was observed by Schleifer and Mansfield. 25 So cAMP accumulation in the presence of IBMX was not necessarily related to a PGE2 effect. This is in agreement with the data of Piguet-Pellorce and Dy 2 who reported that the increase in histamine release by bone marrow cells stimulated by LPS plus GM-CSF, was related to an increase of cAMP, which is not related to PGE2 synthesis. In contrast, PGE2 can increase NO production by LPS-stimulated Kuppfer cells. 27 In IL-2 or IL-2 + LPS cultures, the presence of IBMX reduced NO production, although the inhibition was less than in LPS culture; however, IFN production was drastically reduced. On the other hand PGE2 induced a slight but significant reduction of NO in IL-2 cultures, but we did not observe a PGE2 effect in LPS + IL-2 cultures. This suggests that IL-2 or IL-2 + LPS induce the release of factors which counterbalance the inhibitory effect of PGE2. Moreover IL-2 has been reported to partly reverse the inhibitory effect of PGE2.28 The signal transduction pathway by which IFN-3' induces NO production remains to be established. PKC is involved in the induction of nitric oxide synthase by IFN-3,, protein kinase C activity is increased by IFN-, but not by IFN-cz/]3 or LPS. 29 Work by Fujihara et a/. 3 showed that PKC plays an important role in the LPS-triggered signal transduction pathway. However, other different protein kinases such as PKA via PGE2 induction may be involved. There is also a link between PKA-mediated signalling and nitric oxide synthase. 11 Using H-7, an inhibitor of PKC, we observed a similar reduction of NO that is induced by LPS or IL-2. No effect was observed when the cells were cultured in the presence of IL-2 + LPS, but the presence of H-7 induced a large reduction (97%) of IFN induced by LPS + IL-2. Gessani et al. 3 showed that treatment of macrophages by staurosporine, an inhibitor of PKC, stimulated by IFN-3t inhibits IFN secretion.
There is a relationship between intracellular cAMP concentration and PKC activation. 32 H-7 was an inhibitor of PKC and also of PKA. One might hypothesize that the inhibition of NO induced by H-7 may correspond to the PKA inhibition of LPS, and the PKC inhibition of IL-2.
H-7 did not affect NO production in LPS + IL-2 culture but reduced IFN production. It must be emphasized that inhibition of PKC may involve the reduction of the inhibitors of NO production such as TGF- 33'34 and/or IL-4. 5' On the other hand, as we have shown, it may be that IFN-cz/J3 plays an inhibitory role in NO production, and the fall of IFN-0t/[3 might explain the maintainance of NO levels.
We underline that two metabolic inhibitors, i.e. IBMX and H-7, which did not affect the NO level, reduced IFN production in IL-2 + LPS cultures, whereas N-MMA, which inhibited NO production, increased IFN levels.