Plasma concentrations of alpha-melanocyte-stimulating hormone are elevated in patients on chronic haemodialysis.

BACKGROUND: Clinical and/or laboratory signs of systemic inflammation occur frequently in patients undergoing long-term haemodialysis. It is likely, therefore, that a compensatory release of endogenous anti-inflammatory molecules occurs to limit host reactions. The aim of the present research was to determine if the potent anti-inflammatory peptide alpha-melanocyte-stimulating hormone (alpha-MSH), a pro-opiomelanocortin derivative, is increased in plasma of haemodialysis patients. Because endotoxin and cytokines induce alpha-MSH in vivo and in vitro, we also measured plasma concentrations of endotoxin, interleukin-6 (IL-6), and tumour necrosis factor alpha (TNF-alpha), and the two circulating products of activated monocytes, nitric oxide (NO) and neopterin. METHODS: Thirty-five chronic haemodialysis patients, 20 patients with chronic renal failure not yet on dialysis, and 35 normal controls were included in the study. In the haemodialysis group, blood samples were obtained before and at the end of a dialysis session. Plasma alpha-MSH was measured using a double antibody radioimmunoassay, and IL-6, TNF-alpha, and neopterin using specific enzyme-linked immunosorbent assays. Plasma nitrites were determined by a colorimetric method, and endotoxin with the quantitative chromogenic LAL (limulus amoebocyte lysate) method. RESULTS: Mean plasma alpha-MSH was higher in haemodialysis patients than in control subjects, with the peptide concentrations being particularly elevated in dialysed patients with detectable endotoxin. High alpha-MSH concentrations were observed in the pre-dialysis samples, with no substantial change at the end of the dialysis session. Plasma concentrations of IL-6, TNF-alpha, neopterin, and NO were generally elevated in chronic haemodialysis patients and there was a negative correlation between circulating alpha-MSH and IL-6. In patients with renal failure not yet on dialysis, mean plasma alpha-MSH was similar to that of normal subjects. CONCLUSIONS: alpha-MSH is increased in the circulation of chronic haemodialysis patients and particularly so in case of detectable endotoxaemia. Reduction of renal clearance is unlikely to contribute to the observed rise of the peptide because alpha-MSH concentration is not increased in patients with chronic renal failure who are not yet on dialysis. It is likely that dialysis-associated endotoxaemia, directly and/or through cytokine release, enhances the production of the anti-inflammatory mediator alpha-MSH that limits host reactions.

alpha-MSH and its receptors in regulation of tumor necrosis factor-alpha production by human monocyte/macrophages.

The hypothesis that macrophages contain an autocrine circuit based on melanocortin [ACTH and alpha-melanocyte-stimulating hormone (alpha-MSH)] peptides has major implications for neuroimmunomodulation research and inflammation therapy. To test this hypothesis, cells of the THP-1 human monocyte/macrophage line were stimulated with lipopolysaccharide (LPS) in the presence and absence of alpha-MSH. The inflammatory cytokine tumor necrosis factor (TNF)-alpha was inhibited in relation to alpha-MSH concentration. Similar inhibitory effects on TNF-alpha were observed with ACTH peptides that contain the alpha-MSH amino acid sequence and act on melanocortin receptors. Nuclease protection assays indicated that expression of the human melanocortin-1 receptor subtype (hMC-1R) occurs in THP-1 cells; Southern blots of RT-PCR product revealed that additional subtypes, hMC-3R and hMC-5R, also occur. Incubation of resting macrophages with antibody to hMC-1R increased TNF-alpha concentration; the antibody also markedly reduced the inhibitory influence of alpha-MSH on TNF-alpha in macrophages treated with LPS. These results in cells known to produce alpha-MSH at rest and to increase secretion of the peptide when challenged are consistent with an endogenous regulatory circuit based on melanocortin peptides and their receptors. Targeting of this neuroimmunomodulatory circuit in inflammatory diseases in which myelomonocytic cells are prominent should be beneficial.

Inhibition of systemic inflammation by central action of the neuropeptide alpha-melanocyte- stimulating hormone.

The neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) reduces fever and acute inflammation in the skin when administered centrally. The aim of the present research was to determine whether central alpha-MSH can also reduce signs of systemic inflammation in mice with endotoxemia. Increases in serum tumor necrosis factor-alpha and nitric oxide, induced by intraperitoneal administration of endotoxin, were modulated by central injection of a small concentration of alpha-MSH. Inducible nitric oxide synthase (iNOS) activity and iNOS mRNA in lungs and liver were likewise modulated by central alpha-MSH. Lung myeloperoxidase activity, a marker of neutrophil infiltration, was increased in endotoxemic mice; the increase was significantly less in lungs of mice treated with central alpha-MSH. Intraperitoneal administration of the small dose of alpha-MSH that was effective centrally did not alter any of the markers of inflammation. In experiments using immunoneutralization of central alpha-MSH, we tested the idea that endogenous peptide induced within the brain during systemic inflammation modulates host responses to endotoxic challenge in peripheral tissues. The data showed that proinflammatory agents induced by endotoxin in the circulation, lungs, and liver were significantly greater after blockade of central alpha-MSH. The results suggest that anti-inflammatory influences of neural origin that are triggered by alpha-MSH could be used to treat systemic inflammation.

alpha-MSH in systemic inflammation. Central and peripheral actions.

Until recently, inflammation was believed to arise from events taking place exclusively in the periphery. However, it is now clear that central neurogenic influences can either enhance or modulate peripheral inflammation. Therefore, it should be possible to improve treatment of inflammation by use of antiinflammatory agents that reduce peripheral host responses and inhibit proinflammatory signals in the central nervous system (CNS). One such strategy could be based on alpha-melanocyte stimulating hormone (alpha-MSH). Increases in circulating TNF-alpha and nitric oxide (NO), induced by intraperitoneal administration of endotoxin in mice, were modulated by central injection of a small concentration of alpha-MSH. Inducible nitric oxide synthase (iNOS) activity and iNOS mRNA in lungs and liver were likewise modulated by central alpha-MSH. Increase in lung myeloperoxidase (MPO) activity was significantly less in lungs of mice treated with central alpha-MSH. Proinflammatory agents induced by endotoxin were significantly greater after blockade of central alpha-MSH. The results suggest that antiinflammatory influences of neural origin that are triggered by alpha-MSH could be used to treat systemic inflammation. In addition to its central influences, alpha-MSH has inhibitory effects on peripheral host cells, in which it reduces release of proinflammatory mediators. alpha-MSH reduces chemotaxis of human neutrophils and production of TNF-alpha, neopterin, and NO by monocytes. In research on septic patients, alpha-MSH inhibited release of TNF-alpha, interleukin-1 beta (IL-1 beta), and interleukin-8 (IL-8) in whole blood samples in vitro. Combined central and peripheral influences can be beneficial in treatment of sepsis.

Protective effect of ciliary neurotrophic factor (CNTF) in a model of endotoxic shock: action mechanisms and role of CNTF receptor alpha.

Ciliary neurotrophic factor (CNTF) inhibits the production of tumor necrosis factor (TNF) in lipopolysaccharide (LPS)-treated mice and protects against LPS lethality when coadministered with its soluble receptor (sCNTFR alpha). Both of these activities are abolished in adrenalectomized (ADX) mice. LPS-induced pulmonary polymorphonuclear neutrophil (PMN) infiltration and nitric oxide (NO) production were also inhibited by CNTF + sCNTFR alpha but not by CNTF alone. sCNTFR alpha did not alter the clearance or tissue distribution of CNTF. Furthermore, CNTF variants coadministered with sCNTFR alpha protected against LPS toxicity in a manner related to their affinity for the beta components of CNTFR. Thus, inhibition of TNF production and protection against LPS lethality by CNTF/sCNTFR alpha require an intact hypothalamus-pituitary-adrenal axis (HPAA) and may be mediated by endogenous glucocorticoids. This protective effect is, at least in part, due to the inhibition of PMN infiltration and NO production, and appears to be mediated by cells displaying only beta-receptor subtypes.

Melanocortin peptides inhibit production of proinflammatory cytokines and nitric oxide by activated microglia.

Inflammatory processes contribute to neurodegenerative disease, stroke, encephalitis, and other central nervous system (CNS) disorders. Activated microglia are a source of cytokines and other inflammatory agents within the CNS and it is therefore important to control glial function in order to preserve neural cells. Melanocortin peptides are pro-opiomelanocortin-derived amino acid sequences that include alpha-melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropic hormone (ACTH). These peptides have potent and broad anti-inflammatory effects. We tested effects of alpha-MSH (1-13), alpha-MSH (11-13), and ACTH (1-24) on production of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and nitric oxide (NO) in a cultured murine microglial cell line (N9) stimulated with lipopolysaccharide (LPS) plus interferon gamma (IFN-gamma). Melanocortin peptides inhibited production of these cytokines and NO in a concentration-related fashion, probably by increasing intracellular cAMP. When stimulated with LPS + IFN-gamma, microglia increased release of alpha-MSH. Production of TNF-alpha, IL-6, and NO was greater in activated microglia after innmunoneutralization of endogenous alpha-MSH. The results suggest that alpha-MSH is an autocrine factor in microglia. Because melanocortin peptides inhibit production of pro-inflammatory mediators by activated microglia they might be useful in treatment of inflammatory/degenerative brain disorders.

Effects of methyl palmitate on cytokine release, liver injury and survival in mice with sepsis.

The effects of methyl palmitate (MP), a known inhibitor of Kupffer cells, were studied in a model of polymicrobial sepsis induced in CD-1 mice by cecal ligation and puncture (CLP). The inhibition of Kupffer cells by pretreatment with MP was shown by the reduced phagocytosis, the production of tumor necrosis factor (TNF) and interleukin-6 (IL-6) after lipopolysaccharide (LPS) challenge. The reduced activation of Kupffer cells resulted in lower levels of inflammatory products after CLP. TNF and IL-6 were significantly reduced in serum 2 h and 24 h respectively after CLP, interleukin-1 beta (IL-1 beta) was reduced in liver 4 h after CLP, nitric oxide (NO) and serum amyloid A (SAA) were significantly reduced 8 and 24 h respectively after CLP. Liver toxicity was significantly reduced in MP-treated mice and survival was significantly prolonged at all intervals, reaching 45% after six to ten days compared with 3% in control mice. These findings suggest that Kupffer cells play an important role in liver damage and survival in sepsis.

Chlorpromazine inhibits tumour necrosis factor synthesis and cytotoxicity in vitro.

Chlorpromazine (CPZ) has been previously shown to protect against endotoxin [lipopolysaccharide (LPS)] lethality and inhibit the release of tumour necrosis factor in vivo. We investigated at the cellular level whether this was due to direct inhibition of tumour necrosis factor-alpha (TNF-alpha) synthesis, using LPS-stimulated THP-1 human monocytic leukemia cells. We also studied the effect of CPZ on human TNF-alpha action by assessing TNF-alpha cytotoxicity on mouse fibrosarcoma L929 cells. CPZ (1-100 microM) inhibited TNF-alpha production in THP-1 cells in a dose dependent manner by a maximum of 80%. This effect was comparable to that of two well-known inhibitory drugs, dexamethasone and cyclicAMP. Inhibition was also evident at the mRNA level. On the other hand CPZ (10-25 microM) also inhibited TNF-alpha activity: in fact it reduced the cytotoxicity of TNF-alpha on L929 cells (EC50 was increased four times) and could provide protection even as a post-treatment. CPZ inhibited TNF-induced apoptosis in L929 cells, as detected by analysis of nuclear morphology. However, since we showed that apoptosis was very limited, and was not the main mode of cell death in our conditions, this could not explain the overall protection. Since CPZ did not interfere with either the oligomerization state of TNF-alpha or its receptor binding, our data suggest that it reduced cytotoxicity by inhibiting some steps in the TNF-alpha signalling pathways.

Ciliary neurotrophic factor inhibits brain and peripheral tumor necrosis factor production and, when coadministered with its soluble receptor, protects mice from lipopolysaccharide toxicity.

BACKGROUND: The receptor of ciliary neurotrophic factor (CNTF) contains the signal transduction protein gp130, which is also a component of the receptors of cytokines such as interleukin (IL)-6, leukemia-inhibitory factor (LIF), IL-11, and oncostatin M. This suggests that these cytokines might share common signaling pathways. We previously reported that CNTF augments the levels of corticosterone (CS) and of IL-6 induced by IL-1 and induces the production of the acute-phase protein serum amyloid A (SAA). Since the elevation of serum CS is an important feedback mechanism to limit the synthesis of proinflammatory cytokines, particularly tumor necrosis factor (TNF), we have investigated the effect of CNTF on both TNF production and lipopolysaccharide (LPS) toxicity. MATERIALS AND METHODS: To induce serum TNF levels, LPS was administered to mice at 30 mg/kg i.p. and CNTF was administered as a single dose of 10 micrograms/mouse i.v., either alone or in combination with its soluble receptor sCNTFR alpha at 20 micrograms/mouse. Serum TNF levels were the measured by cytotoxicity on L929 cells. In order to measure the effects of CNTF on LPS-induced TNF production in the brain, mice were injected intracerebroventricularly (i.c.v.) with 2.5 micrograms/kg LPS. Mouse spleen cells cultured for 4 hr with 1 microgram LPS/ml, with or without 10 micrograms CNTF/ml, were also analyzed for TNF production. RESULTS: CNTF, administered either alone or in combination with its soluble receptor, inhibited the induction of serum TNF levels by LPS. This inhibition was also observed in the brain when CNTF and LPS were administered centrally. In vitro, CNTF only marginally affected TNF production by LPS-stimulated mouse splenocytes, but it acted synergistically with dexamethasone (DEX) in inhibiting TNF production. Most importantly, CNTF administered together with sCNTFR alpha protected mice against LPS-induced mortality. CONCLUSIONS: These data suggest that CNTF might act as a protective cytokine against TNF-mediated pathologies both in the brain and in the periphery.

Protective effect of chlorpromazine on TNF-mediated hapten-induced irritant reaction.

Picryl chloride-induced irritant reaction (IR) was shown to be mediated by tumor necrosis factor (TNF). Anti-TNF monoclonal antibodies, but not interleukin 1 receptor antagonist (IL-1 Ra), had a protective effect. Chlorpromazine (CPZ), an inhibitor of TNF synthesis, protected against IR and inhibited the IR-associated TNF induction in ear homogenates. Investigation of the role of polymorphonuclear leukocyte (PMN) in neutropenic mice showed that neutropenia did not prevent the development of the IR.

Mechanisms of interleukin-2-induced hydrothoraxy in mice: protective effect of endotoxin tolerance and dexamethasone and possible role of reactive oxygen intermediates.

Interleukin (IL)-2 is known to induce vascular leak syndrome (VLS), which was suggested to be mediated by immune system-derived cytokines, including tumor necrosis factor (TNF). To characterize the role of TNF in IL-2 toxicity in C3H/HeN mice, we used two approaches to downregulate TNF production in vivo: treatment with dexamethasone (DEX) and induction of endotoxin (lipopolysaccharide) (LPS) tolerance by a 4-day pretreatment with LPS (35 micrograms/mouse/day). Mice were then treated with IL-2 for 5 days (1.8 x 10(5) IU/mouse, twice daily). Both DEX and LPS tolerance blocked development of hydrothorax in IL-2-treated mice and inhibited TNF induction. DEX and LPS tolerance also ameliorated IL-2 toxicity in terms of decrease in food intake and inhibited the increase of the acute-phase protein, serum amyloid A (SAA). The IL-2 activation of splenic natural killer (NK) cell activity was also diminished by DEX and, to a lesser extent, by LPS-tolerance. Treatment with IL-2 also caused induction of the superoxide-generating enzyme xanthine oxidase (XO) in tissues and serum and induced bacterial translocation in the mesenteric lymph nodes (MLN). These data suggest that multiple mechanisms, including NK cell activity, cytokines, and reactive oxygen intermediates, might be important in the vascular toxicity of IL-2.

Differential effect of glucocorticoids on tumour necrosis factor production in mice: up-regulation by early pretreatment with dexamethasone.

Glucocorticoids (GC) are well known inhibitors of tumour necrosis factor (TNF) production. We investigated the role of endogenous GC in the regulation of TNF production in mice treated with lipopolysaccharide (LPS) using a pretreatment with dexamethasone (DEX) to down-regulate the hypothalamus-pituitary-adrenal axis (HPA). Short-term DEX pretreatment (up to 12 h before LPS) inhibited TNF production, but earlier (24-48 h) pretreatments potentiated it. This up-regulating effect was not observed in adrenalectomized mice or when GC synthesis was inhibited with cyanoketone (CK). This effect could not be explained only by the suppression of LPS-induced corticosterone (CS) levels induced by DEX, since a 48-h pretreatment potentiated TNF production without affecting LPS-induced CS levels. On the other hand, mice chronically pretreated with DEX were still responsive to its inhibitory effect on TNF production, thus ruling out the possibility of a decreased responsiveness to GC.

Role of xanthine oxidase and reactive oxygen intermediates in LPS- and TNF-induced pulmonary edema.

We studied the role of reactive oxygen intermediates (ROI) in lipopolysaccharide (LPS)-induced pulmonary edema. LPS treatment (600 micrograms/mouse, IP) was associated with a marked induction of the superoxide-generating enzyme xanthine oxidase (XO) in serum and lung. Pretreatment with the antioxidant N-acetylcysteine (NAC)--1 gm/kg orally, 45 minutes before LPS--or with the XO inhibitor allopurinol (AP)--50 mg/kg orally at -1 hour and +3 hours--was protective. On the other hand nonsteroidal antiinflammatory drugs (ibuprofen, indomethacin, and nordihydroguaiaretic acid) were ineffective. These data suggested that XO might be involved in the induction of pulmonary damage by LPS. However, treatment with the interferon inducer polyriboinosylic-polyribocytidylic acid, although inducing XO to the same extent as LPS, did not cause any pulmonary edema, indicating that XO is not sufficient for this toxicity of LPS. To define the possible role of cytokines, we studied the effect of direct administration of LPS (600 micrograms/mouse, IP), tumor necrosis factor (TNF, 2.5 or 50 micrograms/mouse, IV), interleukin-1 (IL-1 beta, 2.5 micrograms/mouse, IV), interferon-gamma (IFN-gamma, 2.5 micrograms/mouse, IV), or their combination at 2.5 micrograms each. In addition to LPS, only TNF at the highest dose induced pulmonary edema 24 hours later. LPS-induced pulmonary edema was partially inhibited by anti-IFN-gamma antibodies but not by anti-TNF antibodies, anti-IL-1 beta antibodies, or IL-1 receptor antagonist (IL-1Ra).