Muramyl-dipeptide-induced mitochondrial proton leak in macrophages is associated with upregulation of uncoupling protein 2 and the production of reactive oxygen and reactive nitrogen species.

The synthetic immunomodulator muramyl dipeptide (MDP) has been shown to induce, in vivo, mitochondrial proton leak. In the present work, we extended these findings to the cellular level and confirmed the effects of MDP in vitro on murine macrophages. The macrophage system was then used to analyse the mechanism of the MDP-induced mitochondrial proton leak. Our results demonstrate that the cellular levels of superoxide anion and nitric oxide were significantly elevated in response to MDP. Moreover, isolated mitochondria from cells treated with MDP presented a significant decrease in respiratory control ratio, an effect that was absent following treatment with a non-toxic analogue such as murabutide. Stimulation of cells with MDP, but not with murabutide, rapidly upregulates the expression of the mitochondrial protein uncoupling protein 2 (UCP2), and pretreatment with vitamin E attenuates upregulation of UCP2. These findings suggest that the MDP-induced reactive species upregulate UCP2 expression in order to counteract the effects of MDP on mitochondrial respiratory efficiency.

Nitric oxide up-regulates DNA-binding activity of nuclear factor-kappaB in macrophages stimulated with silica and inflammatory stimulants.

Nitric oxide (NO), a reactive nitrogen species, plays an important role in inflammatory lung damage. In the present study, we investigated the role of NO in DNA-binding activity of NF-kappaB in macrophages stimulated with silica or other inflammatory stimulants. Treatment of mouse macrophages (RAW264.7 cells) with a selective inhibitor of inducible nitric oxide synthase (iNOS), L-N6-(1-iminoethyl) lysine (L-NIL), or a nonselective iNOS inhibitor, N omega-nitro-L-arginine methylester (L-NAME), resulted in inhibition of silica-induced nitric oxide production as well as silica-induced NF-kappaB activation. L-NIL also effectively inhibited NF-kappaB activation induced by other inflammatory stimulants, such as lipopolysaccharide (LPS) or muramyl dipeptide (MDP). These inhibitory effects of L-NIL and L-NAME on silica- or LPS-induced NF-kappaB activation were also observed in primary rat alveolar macrophages. Furthermore, NO generating compounds, such as sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), caused a dose-dependent increase in NF-kappaB activation, which was positively correlated with the level of NO production. Specific inhibitors of protein tyrosine kinase, such as genistein and AG494, prevented NF-kappaB activation in SNP- or SIN-1 treated cells, suggesting involvement of tyrosine kinase in the NO signaling pathway leading to NF-kappaB activation. In contrast, inhibitors of protein kinase C or A, such as staurosporine or H89, had no inhibitory effect on SIN-1 induced NF-kappaB activation. Metalloporphyrins, such as tetrakis (N-methyl-4'-pyridyl) porphyrinato iron (III) (Fe-TMPyP) and Zn-TMPyP which are known to alter NO-dependent activity, markedly inhibited silica- and LPS-induced NF-kappaB activation. The results suggest that NF-kappaB activation in macrophages can be induced under certain conditions by nitric oxide and that nitric oxide produced by phagocytes exposed to inflammatory agents may up-regulate the activation of NF-kappaB.

Liposomal phosphatidylserine inhibits tumor cytotoxicity of liver macrophages induced by muramyl dipeptide and lipopolysaccharide.

Liposomes can very efficiently deliver immunomodulators to macrophages so as to induce tumor cytotoxicity. Liposomes most widely used for that purpose contain negatively charged lipids, in particular phosphatidylserine (PS), to enhance liposome uptake by the macrophages. We investigated the effect of three negatively charged liposomal lipids on the in vitro activation of liver macrophages to tumor cytotoxicity by muramyl dipeptide (MDP) and lipopolysaccharide (LPS). Both MDP- and LPS-induced tumor cytotoxicity towards murine colon adenocarcinoma cells were strongly inhibited by PS-containing liposomes. Under comparable conditions phosphatidylglycerol (DPPG)-containing or dicetyl phosphate (DCP)-containing liposomes did not inhibit or only marginally inhibited the induction of tumor cytotoxicity. We did not observe PS-mediated inhibition of tumor cell toxicity when the exposure of the macrophages to PS-liposomes was limited to the 4-h activation period prior to addition of the tumor target cells, suggesting that the inhibitory effect is accomplished at the level of the later stages of the activation process. Previously, we showed that macrophages which are activated to tumor cytotoxicity during a 24-h incubation with MDP become refractory to a second activation with MDP. Now we observed that simultaneous incubation with PS-containing liposomes partially prevents this refractoriness, which is also compatible with an interfering action of PS at a relatively late stage in the activation process. We conclude that PS, despite its reported stimulatory effect on liposome uptake by macrophages, can seriously antagonize the effectiveness of immunomodulating agents acting on macrophages. This bears relevance to the use of PS-containing liposomes as a vehicle for such agents. The results are discussed in perspective of earlier reported pharmacological effects of PS and its metabolites.

Bacterial products and the control of ingestive behavior: clinical implications.

Bacterial products such as lipopolysaccharides (LPS) and muramyl peptides are delivered in the course of infections. They trigger the host's acute phase responses to bacterial infections and are probably involved in the accompanying hypophagia because LPS and muramyl dipeptide (MDP, the minimal immunologically active muramyl peptide) reduce food intake after parenteral administration in animals. LPS and MDP inhibit feeding synergistically through separate but interacting mechanisms. The hypophagic effects of LPS and MDP are presumably mediated by the combined actions of interleukin-1, tumor necrosis factor, and other cytokines. More work is required to understand the interactions between these cytokines, and between bacterial products and cytokines, before cytokine antagonists can be used for treatment of the hypophagia during bacterial infections. As the hypophagia seems to be an early mechanism of host defense, a treatment should be carefully considered. If an intervention is indicated because of a patient's poor condition, inhibitors of eicosanoid synthesis and glucocorticoids may hold more promise for therapy because such substances block LPS and MDP hypophagia. Although LPS can reduce food intake by direct action on the brain, presently available evidence indicates that systemic LPS acts primarily in the periphery to generate a neural signal that is transmitted to the brain and inhibits feeding through the vagus. The exact site where LPS acts on peripheral nerves remains to be identified. LPS hypophagia is conditionable, but conditioning cannot solely account for LPS hypophagia under most test conditions. Whether MDP hypophagia is also conditionable and mediated by vagal afferents is not yet known. All in all, the putative mediators and mechanisms of LPS and MDP hypophagia suggest some options for a treatment of the hypophagia during bacterial infection, but present knowledge about the mechanisms and interactions of the involved substances is still fragmentary and requires further investigation.

An IL-1 receptor and an IL-1 receptor antagonist attenuate muramyl dipeptide- and IL-1-induced sleep and fever.

It is hypothesized that the somnogenic and pyrogenic effects of muramyl dipeptide (MDP) are mediated via enhanced interleukin-1 (IL-1) production. To test this hypothesis the effects of intracerebroventricular (icv) administration of a recombinant human soluble type I IL-1 receptor (sIL-1r) and of the IL-1 receptor antagonist (IL-1ra) on MDP-induced sleep and fever were evaluated in rabbits. The sIL-1r recognized rabbit IL-1 beta, but it did not affect sleep or brain temperature across the dose range tested (1-50 micrograms) when injected icv into normal rabbits. Pretreatment of rabbits with 50 micrograms sIL-1r or 10 micrograms IL-1ra blocked human recombinant IL-1-enhanced nonrapid eye movement (NREM) sleep and fever. Thus both the sIL-1r and the IL-1ra were effective antagonists of IL-1 actions. When the animals were pretreated with either 50 micrograms sIL-1r or with 10 or 100 micrograms of the IL-1ra, the somnogenic effects of 150 pmol MDP were attenuated. However, the sIL-1r had little effect on MDP-induced febrile responses. These results suggest that the sIL-1r and the IL-1ra can function as antagonists of IL-1 actions in vivo and that MDP-induced sleep and fever are partially mediated by IL-1.

Pharmacological aspects of arthritis induced by a muramyl dipeptide analogue in rats.

Fourteen consecutive daily subcutaneous injections of 4 mg/kg of the muramyl dipeptide analogue MDP-Lys(L18) into rats caused arthritis characterized by swelling of the tarsal joint, increases in lymphocytes and monocytes in the peripheral blood, and elevated serum immunoglobulin G (IgG). The present study was performed to evaluate the effects of indomethacin, phenylbutazone, dexamethasone, D-penicillamine, aurothioglucose, cyclophosphamide and cyclosporin A on this arthritis. Administration of indomethacin, phenylbutazone or dexamethasone inhibited the development of the tarsal joint swelling, suggesting that prostaglandins may be involved in the pathogenesis of the arthritis. Cyclophosphamide reduced the arthritis, together with decreases in the lymphocyte count and the serum IgG level. Cyclosporin A worsened the arthritis in a dose-dependent manner and increased the neutrophil count without raising the serum IgG level, but inhibited the induction of adjuvant arthritis in rats with Mycobacterium bacilli. MDP-Lys(L18) may therefore induce arthritis differing in mechanism from adjuvant arthritis.

Uveitis induction in the rabbit by muramyl dipeptides.

Intraocular inflammation (uveitis) was produced in rabbits by intravenous or subcutaneous treatment with N-acetylmuramyl-L-alanyl-D-isoglutamine and several of its synthetic analogs at doses of greater than or equal to 0.2 mg/kg in saline. A dose-dependent increase in permeability of the ocular blood-aqueous barrier as measured by leakage of protein or fluoresceinated dextran from the serum into the eye was observed from 2 to 14 h after glycopeptide treatment. Peak response occurred at approximately 3 h postdose. The lowest dose found to produce maximal vascular leakage for the most active glycopeptide analogs was 1 mg/kg. The adjuvant-inactive L-L stereoisomer of N-acetylmuramyl-L-alanyl-D-isoglutamine was inactive, even at doses as high as 10 mg/kg. Analogs of N-acetylmuramyl-L-alanyl-D-isoglutamine which were homologous in the lactyl side chain were found to cause less uveitis. Chronic biweekly intravenous treatment of rabbits for 1 month with either N-acetyl-L-alpha-aminobutyryl-D-isoglutamine or its lipophilic 6-O-stearoyl derivative at 1 mg/kg, but not with murabutide, resulted in leukocytic inflammatory lesions unique to the uveal tract of the eye. The uveitis was potentially reversible and occurred with decreased severity as long as 2 months after cessation of chronic treatment. Vascular leakage but not cellular infiltrate in the choroid could be modulated by pharmacologic means. Pyrogenicity but not adjuvanticity correlated with ability of glycopeptides to induce vascular leakage. Several adjuvant-active muramyl dipeptide analogs with minimal ability to cause acute vascular leakage or chronic inflammation in the rabbit eye have been identified.