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P2Z/P2X7 receptor is a particular type of purinoceptor, which is selectively expressed on the surface of immune cells in neuronal and non-neuronal tissues. Despite intensive research on its involvement in the immune response, the exact mechanism whereby it affects intercellular signaling is far from clear yet. In this study, the effect of activation P2Z/P2X7 receptor was investigated on the bacterial lipopolysaccharide induced nitric oxide production in RAW 264.7 macrophage call line using the nitrite/nitrate assay. The P2Z/P2X7 receptor agonist 3'-O-(4-benzoylbenzoyl)-adenosine 5'triphosphate increased concentration-dependency the lipoplysaccharide (10 microg/ml) induced nitric oxide production between 10 microM and 250 microM. ATP also increased nitric oxide production in response to lipopolysaccharide, while ADP, 2-methylo-thio-adenosine 5'-triphosphate and adenosine 5'triphosphate-gamma-S was without effect. Pretreatment with oxidized adenosine triphosphate, the selective P2Z/P2X7 receptor antagonise (300 microM-1 microM) strongly decreased lipopolysaccaride induced nitric oxide production. Furthermore, on macrophages, pretreated with oxidized adenosine 5'-triphosphate (300 microM-1 mM), 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate and ATP did not affect lipopolysaccharide induced nitric oxide production. 15 min lipopolysaccharide treatment induced a transient and reversible release of endogenous ATP from RAW 264.7 cells, measured by the luciferin-luciferase assay. The effect of lipopolysaccharide to promote ATP release was concentration-dependent between 1-10 microg/ml. In summary, our results show that P2Z/P2X7 receptor activation results in an increase in nitric oxide production in response to lipopolysaccharide challenge. Since the P2Z/P2X7 receptor antagonist oxidized adenosine triphosphate decreased lipopolysaccharide induced nitric oxide production, and lipopolysaccharide was able to promote ATP release from macrophage cells, it seems likely that endogenous ATP is involved in nitric oxide formation during endotoxin challenge.

During endotoxemia, immune cells activated by lipopolysaccharide (LPS) produce various inflammatory mediators, including cytokines and nitric oxide (NO). The genes of several mediators are activated in part by the rapid binding of the transcription factor nuclear factor-kappa B (NF-kappaB) to its promoter. The induction of this transcription factor can be blocked by a wide range of antioxidants, including pyrrolidine dithiocarbamate (PDTC). Here we investigated in mice the effect of this compound on the plasma tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-1alpha (IL-1alpha), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), macrophage inflammatory protein-1alpha (MIP-1alpha), and nitric oxide (NO) response to intraperitoneal (i.p.) injection of LPS. Pretreatment of animals with PDTC (10-100 mg/kg) 30 min prior to LPS challenge (4 mg/kg, i.p.) decreased plasma TNF-alpha, IL-12, MIP-1alpha, and nitrite/nitrate (breakdown products of NO) concentrations, but enhanced plasma levels of IL-10. Moreover, pretreatment of mice with PDTC (10-100 mg/kg, i.p.) did not alter LPS-induced (4 mg/kg) production of IL-1alpha, IL-6, and IFN-gamma. Finally, PDTC (100 mg/kg) protected the mice against LPS (100 mg/kg)-induced lethality. These results indicate that blockade of the NF-kappaB pathway by PDTC has potent anti-inflammatory action in systemic inflammatory processes.

1. Intracellular calcium has been suggested to be an important mediator of the cellular response in endotoxaemia and shock. Dantrolene is an agent that interferes with intracellular calcium fluxes resulting in a decreased availability of calcium in the cytoplasm. Here we have investigated the effect of dantrolene on lipopolysaccharide (LPS)-induced production of interleukin-10 (IL-10), tumour necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) in mice and in cultured RAW 264.7 macrophages in vitro. 2. In BALB/c mice, LPS-induced plasma IL-10 levels were significantly enhanced by pretreatment with dantrolene (20 mg kg(-1), i.p.) (P < 0.005 at the 90 min time-point). On the other hand, dantrolene pretreatment suppressed circulating TNF-alpha and nitrite/nitrate (breakdown products of NO) concentrations. However, dantrolene had no effect on LPS-induced plasma interleukin-6 (IL-6) levels (67.22+/-5.51 ng ml(-1) in vehicle-pretreated mice and 62.22+/-3.66 ng ml(-1) in dantrolene-pretreated mice, n = 9). 3. Dantrolene inhibited TNF-alpha and NO production in C57BL/6 IL-10+/+ mice, as well as in their IL-10 deficient counterparts (C57BL/6 IL-10(0/0)). 4. In RAW 264.7 macrophages, dantrolene (10-300 microM) reduced IL-10, TNF-alpha, and nitrite (breakdown product of NO) production elicited by LPS (10 microg ml(-1)). Dantrolene (300 microM) did not affect the LPS-induced nuclear translocation of transcription factor nuclear factor kappaB in these cells. 5. Although LPS failed to alter the intracellullar concentration of calcium in single macrophages loaded with Fura-2, dantrolene caused a significant decrease of the basal calcium level as determined 30 min after dantrolene treatment (P < 0.005). ATP (1 mM) caused a rapid rise in intracellular calcium levels in both dantrolene-pretreated and vehicle-pretreated cells. 6. These results indicate that unlike the secretion of TNF-alpha and NO, IL-10 production is differentially regulated in vitro and in vivo. The decrease of plasma levels of the pro-inflammatory mediators TNF-alpha and NO, and increase in circulating IL-10 concentrations by dantrolene suggest that this drug might offer a new therapeutic approach in inflammatory diseases and septic shock.

In a previous study we have demonstrated in conscious endotoxemic mice that isoproterenol, a nonselective agonist of beta-adrenergic receptors, decreased the production of proinflammatory mediators tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO), and enhanced the formation of the anti-inflammatory cytokine interleukin-10 (IL-10). In the present study we investigated the effect of isoproterenol on the bacterial lipopolysaccharide (endotoxin, LPS; 10 microg/ml)-induced inflammatory response in RAW 264.7 macrophages in vitro. Pretreatment of cells with isoproterenol (10-300 microM) resulted in an inhibition of TNF-alpha, NO (reflected as its stable breakdown product nitrite), as well as IL-10 production that was paralleled with a restoration of the LPS-induced suppression of mitochondrial respiration. In addition, isoproterenol elevated cAMP accumulation in these cells. Finally, isoproterenol (300 microM) did not influence the nuclear translocation of nuclear factor kappaB. These data demonstrate that isoproterenol potently downregulates the LPS-induced inflammatory response and further support the notion that stimulation of beta-adrenoreceptors can be an effective strategy in the treatment of inflammatory diseases.

Intracellular cyclic nucleotide levels play an important role in the regulation of several immunological processes. Since elevation of intracellular cyclic adenosine monophosphate and/or cyclic guanosine monophosphate concentration by inhibition of phosphodiesterase (PDE) is known to modulate the inflammatory response, we compared the effect of amrinone, an inhibitor of the PDE III isoenzyme, and of theophylline, a nonspecific PDE inhibitor, on the plasma tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-10 (IL-10), and nitric oxide response in mice to intraperitoneal injection of bacterial lipopolysaccharide (LPS). Intraperitoneal treatment of animals with amrinone (100 mg/kg) 30 min before LPS administration decreased both plasma IL-6 and IL-10 concentrations in the first phase of the response, but enhanced plasma levels of these cytokines in the second part. In contrast, pretreatment of the animals with theophylline (100 mg/kg) enhanced LPS-induced plasma IL-6 and IL-10 levels during the whole response. However, pretreatment with both PDE inhibitors resulted in a marked inhibition of LPS-evoked plasma concentrations of TNF-alpha and nitrite/nitrate (breakdown products of nitric oxide) throughout the response. This study demonstrates for the first time that amrinone and theophylline possess differential, but primarily anti-inflammatory, properties during LPS-induced systemic inflammation in the mouse.

Pro-inflammatory cytokines, such as tumour necrosis factor (TNF) and free radicals, such as nitric oxide (NO), are mediators of endotoxaemia. Catecholamines are in clinical use to treat the haemodynamic consequences of severe septic shock. Beta-adrenergic agonists exert many of their effects by elevation of intracellular cyclic AMP (cAMP) concentration. Cyclic AMP can modulate endotoxin-induced cytokine and NO production. Here we investigate the effect of isoproterenol pretreatment on the cytokine and NO production induced by bacterial lipopolysaccharide (LPS, 4-10 mg/kg). Pretreatment with isoproterenol (10 mg/kg) blunted the LPS-induced TNF response, increased the LPS-induced formation of interleukin-10 and interleukin-6 and reduced the LPS-induced production of NO in conscious mice. In anaesthetized rats, pretreatment with isoproterenol prevented the LPS-induced suppression of vascular contractility to norepinephrine in the thoracic aorta ex vivo. The hyporeactivity is due to expression of the inducible isoform of NO synthase (iNOS) and was restored by in vitro administration of NG-methyl-L-arginine (L-NMA), an inhibitor of NO synthase. However, L-NMA did not alter vascular contractility in control vessels or in rings taken from the LPS-treated rats pretreated with isoproterenol. Our findings suggest that, in addition to its haemodynamic actions, isoproterenol may also exert beneficial effects by modulating the endotoxin-induced inflammatory response.

Adenosine released into the extracellular space by immunologic and nonimmunologic stimuli has been shown to regulate various immune functions. In this study we report that i.p. pretreatment of mice with CGS-21680 HCl (CGS), a selective agonist of A2 adenosine receptors, at 0.2 to 2 mg/kg caused an augmentation of plasma IL-10 levels induced by i.p. injection of LPS, but decreased plasma levels of LPS-induced TNF-alpha. 2-Chloro-N6-cyclopentyladenosine (CCPA), an agonist of A1 adenosine receptors, at 0.5 mg/kg diminished LPS-induced plasma TNF-alpha concentrations, but enhanced LPS-induced IL-10 levels only at the highest dose used (2 mg/kg). The specific A3 adenosine receptor agonist 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-beta- D-ribofuranuronamide, at 0.2 and 0.5 mg/kg potentiated LPS-stimulated IL-10 production and inhibited LPS-induced TNF-alpha production. LPS-induced plasma nitrite and nitrate levels (the breakdown products of nitric oxide (NO)) were suppressed by CGS and CCPA. In the RAW 264.7 macrophage cell line, pretreatment of the cells with both CGS and CCPA inhibited LPS-induced IL-10, TNF-alpha, and NO production, each in a concentration-dependent manner. The inhibitory effect of these drugs on cytokine and NO production was associated with improved mitochondrial respiration. Neither CGS nor CCPA affected the LPS-induced nuclear translocation of transcription factor nuclear factor-kappaB in these cells. These results demonstrate that adenosine receptor stimulation differentially modulates the LPS-induced production of IL-10, TNF-alpha, and NO in vitro and in vivo. The increase in LPS-induced IL-10 production and suppression of LPS-induced TNF-alpha and NO production caused by adenosine receptor activation may explain some of the immunomodulatory actions of adenosine released in excess during inflammatory and/or ischemic insult.