Prodigiosins (Ps) represent a family of naturally occurring red pigments characterized by a common pyrrolylpyrromethene skeleton. Some members of this family have been shown to possess interesting immunosuppressive properties exerted with a novel mechanism of action, different from that of currently used drugs. In fact, Ps inhibit phosphorylation and activation of JAK-3, a cytoplasmic tyrosine kinase associated with a cell surface receptor component called common gamma-chain, which is exclusive of all IL-2 cytokine family receptors. Blocking common gamma-chain transduction activity results in a potent and specific immunosuppressive activity. With respect to the interesting and unexploited immunomodulating properties of this family of compounds we initiated a medicinal chemistry program aimed at finding novel prodigiosin derivatives with improved immunosuppressive activity and lower toxicity. Utilizing an unprecedented and flexible way of assembling the prodigiosin frame, a number of new derivatives have been prepared and tested leading to the choice of 4-benzyloxy-5-[(5-undecyl-2H-pyrrol-2-ylidene)methyl]-2, 2'-bi-1H-pyrrole (PNU-156804, 16) as a lead immunosuppressant.

The heme oxygenase 1 (HO-1) gene is rapidly activated in the liver after lipopolysaccharide (LPS) treatment. Ninety minutes after LPS treatment (0.1 mg/kg, intraperitoneally) hepatic HO-1 messenger RNA (mRNA) of mice was 40 times the control value. To investigate the hepatic cellular source of the increased HO-1 transcript, we treated mice with LPS and galactosamine (700 mg/kg, intraperitoneally), a selective transcriptional inhibitor of hepatocytes. Galactosamine prevented the LPS-mediated increase of HO-1 mRNA in the liver, indicating that hepatocytes are the main cell type in which HO-1 mRNA accumulates after LPS treatment. We then tested in vitro and in vivo the hypothesis that LPS-mediated hepatic accumulation of HO-1 mRNA is caused by intercellular communication between Kupffer cells and hepatocytes. Isolated rat hepatocytes showed an increase in HO-1 mRNA compared with controls after 90 minutes of exposure to a LPS stimulated Kupffer cell-conditioned medium. This suggests that soluble mediators from Kupffer cells were responsible for this effect. To study the role of Kupffer cells in vivo, we treated mice with Kupffer cell-inactivating or -depleting agents and LPS. Gadolinium chloride and liposome-encapsulated dichloromethylene diphosphonate lowered LPS-mediated HO-1 mRNA accumulation (by about 50%); in these groups hepatic levels of interleukin (IL)-1beta were decreased, by more than 75%. Methylpalmitate hardly affected hepatic HO-1 mRNA accumulation or IL-1beta content after LPS treatment. There was no relationship between HO-1 mRNA and serum TNF or IL-6 levels. These results suggest that LPS-mediated hepatic HO-1 mRNA accumulation is a hepatocyte response partly caused by soluble mediators, particularly IL-1beta, released from Kupffer cells.

The production of tumor necrosis factor alpha (TNF-␣), interleukin-1␤ (IL-1␤), and IL-6 and their pharmacomodulation were evaluated in a model of polymicrobial sepsis induced in mice by cecal ligation and puncture (CLP) and were compared with the effects of endotoxin (lipopolysaccharide [LPS]) treatment. LPS levels rose as early as 1 h after CLP and increased further after 2 and 21 h. TNF-␣ was detectable in serum, spleen, liver, and lungs during the first 4 h, with a peak 2 h after CLP. IL-1␤ was measurable in serum after 24 h, and levels increased significantly in spleen and liver 4 and 8 h after CLP. IL-6 levels increased significantly in serum throughout the first 16 h after CLP. These cytokines were detectable after LPS injection, with kinetics similar to those after CLP but at a significantly higher level. To cast more light on the differences between these two animal models of septic shock, we studied the effects of different reference drugs. Pretreatment with dexamethasone (DEX); ibuprofen (IBU), an inhibitor of cyclooxygenase; and N G-nitro-L-arginine, an inhibitor of nitric oxide synthase, significantly reduced survival, while chlorpromazine (CPZ) and TNF did not affect it. Only the antibiotics and pentoxifylline significantly increased survival in mice with CLP. However, CPZ and DEX protected the mice from LPS mortality. On inhibiting TNF-␣ with DEX, CPZ, or pentoxifylline, survival was reduced, unchanged, and increased, respectively, and on increasing TNF-␣ with IBU and TNF, survival was decreased or unchanged, respectively, suggesting that the modulation of this cytokine does not play a significant role in sepsis induced by CLP, unlike treatment with LPS. The negative effects of IBU and N G-nitro-L-arginine suggest a protective role by prostaglandins and nitric oxide in sepsis induced by CLP.

We investigated the pattern of down-regulation of cytokine production in endotoxin (lipopolysaccharide [LPS]) tolerance. A 4-day treatment with LPS (35 ,ug per mouse) was followed by a challenge on day 6 with one more injection of LPS. Circulating tumor necrosis factor (TNF) and interleukin-6 (IL-6) could not be induced (>99%o inhibition) by LPS in LPS-tolerant mice; colony-stimulating factor (CSF) was also down-regulated by more than 95%, whereas interferon (IFN) and IL-1 syntheses were only partially inhibited. To study the mechanism of cytokine down-regulation in tolerance, we attempted to reverse the tolerant state by pretreatment with phorbol 12-myristate 13-acetate (PMA) (4 ,ug per mouse) 10 min before the LPS challenge. PMA completely restored IL-6 production and partially that of CSF. PMA had no effect on IFN production and inhibited the induction of IL-1. TNF production was also not restored by PMA. To investigate the role of endogenously produced cytokines in the development of LPS tolerance, we administered IL-6, TNF, or IL-lc, using the same treatment schedule as that for LPS. Whereas IL-6 had no effect, IL-lae or TNF induced partial tolerance to LPS in terms of inhibition of LPS-stimulated TNF and IL-6 production. However, a full LPS-tolerant state could not be induced by administration of recombinant cytokines, suggesting the existence of additional mechanisms, such as a loss of LPS receptors or changes in release of soluble binding proteins.

Interleukin (IL) 10 inhibits endotoxin (lipopolysaccharide; LPS) induced tumor necrosis factor (TNF) production in vivo and in vitro. In turn, IL-10 is induced by LPS and acts as a negative feedback to limit TNF production. We investigated the effects of IL-10 on brain TNF and IL-1β production induced by a central LPS administration in mice. Because central LPS also induces peripheral TNF, we also measured the serum TNF levels. A single intracere-broventricular injection of murine recombinant IL-10 (75 ng/mouse) simultaneously with LPS (2.5 µg/mouse) almost completely inhibited brain TNF production. The brain IL-1β production was also inhibited, as was the serum concentration of the acute-phase protein serum amyloid A. On the other hand, intracerebroventricular administration of an anti-IL-10 monoclonal antibody (JES5-2A5; 60 µg/mouse) potentiated brain TNF and IL-1β production. Identical results were obtained when the serum TNF levels were measured. IL-10 did not affect the LPS-induced increase of serum corticosterone, the main endogenous inhibitor of TNF production, or the induction of IL-6. These results indicate that LPS-induced IL-10 can act as an important endogenous inhibitor of brain TNF production and suggest an anti-inflammatory role for IL-10 in the central nervous system.