Ana Campa

Is serum amyloid A an endogenous TLR4 agonist

Serum amyloid A (SAA), a classical acute‐phase protein, is produced predominantly by hepatocytes in response to injury, infection, and inflammation. It has been shown that SAA primes leukocytes and induces the expression and release of proinflammatory cytokines. Here, we report that SAA induces NO production by murine peritoneal macrophages. Using specific inhibitors, we showed that NO production was dependent on inducible NO synthase thorough the activation of ERK1/2 and p38 MAPKs. Moreover, SAA activity was decreased after proteolysis but not with polymyxin B, a lipid A antagonist. Finally, we found that NO production was dependent on functional TLR4, a receptor complex associated with innate immunity. Macrophages from C3H/HeJ and C57BL/10ScCr mice lacking a functional TLR4 did not respond to SAA stimulation. In conclusion, our study makes a novel observation that SAA might be an endogenous agonist for the TLR4 complex on macrophages. The contribution of this finding in amplifying innate immunity during the inflammatory process is discussed.

Oxidation of melatonin and its catabolites, N1‐acetyl‐N 2‐formyl‐5‐methoxykynuramine and N1‐acetyl‐5‐methoxykynuramine, by activated leukocytes

Abstract:  N 1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (AFMK) and N1‐acetyl‐5‐methoxykynuramine (AMK), two melatonin catabolites, have been described as potent antioxidants. We aimed to follow the kinetics of AFMK and AMK formation when melatonin is oxidized by phorbol myristate acetate (PMA) and lipopolysaccharide (LPS)‐activated leukocytes. An HPLC‐based method was used for AFMK and AMK determination in neutrophil and peripheral blood mononuclear cell cultures supernatants. Samples were separated isocratically on a C18 reverse‐phase column using acetonitrile/H2O (25:75) as the mobile phase. AFMK was detected by fluorescence (excitation 340 nm and emission 460 nm) and AMK by UV‐VIS absorbance (254 nm). Activation of neutrophils and mononuclear cells with PMA produces larger amounts of AFMK than activation with LPS, probably due to the lower levels of reactive oxygen species formation and myeloperoxidase (MPO) degranulation that occurs when cells are stimulated with LPS. The concentration of AMK found in the supernatant was about 5–10% (from 18‐hr cultures) compared with AFMK. This result may reflect its reactivity. Indeed AMK, but not AFMK, is easily oxidized by activated neutrophils in a MPO and hydrogen peroxide‐dependent reaction. In conclusion, we defined a simple procedure for the determination of AFMK and AMK in biological samples and demonstrated the capacity of leukocytes to oxidize melatonin and AMK.

Low density lipoprotein oxidation by stimulated neutrophils and ferritin.

Low density lipoprotein (LDL) oxidation mediated by phorbol myristate acetate (PMA)- and formylmethionylleucylphenylalanine (FMLP) -stimulated human neutrophils was enhanced by 70% in the presence of ferritin. Iron released from ferritin by the superoxide anion generated in the respiratory burst of stimulated neutrophils is shown to be involved in lipoprotein oxidation. Ascorbate (100 microM), superoxide dismutase (10 micrograms/ml) and uric acid (430 microM) showed inhibitory effects of 30% [corrected], 70% and 50% on LDL oxidation, respectively. Ceruloplasmin (2.7 microM) potentiated LDL oxidation by stimulated neutrophils and ferritin, both alone and in the presence of methionine. Methionine (1 mM) and catalase (30 micrograms/ml) increased LDL oxidation by stimulated neutrophils and ferritin. These data suggest that LDL oxidation by stimulated neutrophils and ferritin may be relevant in inflammation when both neutrophils and ferritin are increased.

Superoxide-dependent oxidation of melatonin by myeloperoxidase.

Myeloperoxidase uses hydrogen peroxide to oxidize numerous substrates to hypohalous acids or reactive free radicals. Here we show that neutrophils oxidize melatonin to N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) in a reaction that is catalyzed by myeloperoxidase. Production of AFMK was highly dependent on superoxide but not hydrogen peroxide. It did not require hypochlorous acid, singlet oxygen, or hydroxyl radical. Purified myeloperoxidase and a superoxide-generating system oxidized melatonin to AFMK and a dimer. The dimer would result from coupling of melatonin radicals. Oxidation of melatonin was partially inhibited by catalase or superoxide dismutase. Formation of AFMK was almost completely eliminated by superoxide dismutase but weakly inhibited by catalase. In contrast, production of melatonin dimer was enhanced by superoxide dismutase and blocked by catalase. We propose that myeloperoxidase uses superoxide to oxidize melatonin by two distinct pathways. One pathway involves the classical peroxidation mechanism in which hydrogen peroxide is used to oxidize melatonin to radicals. Superoxide adds to these radicals to form an unstable peroxide that decays to AFMK. In the other pathway, myeloperoxidase uses superoxide to insert dioxygen into melatonin to form AFMK. This novel activity expands the types of oxidative reactions myeloperoxidase can catalyze. It should be relevant to the way neutrophils use superoxide to kill bacteria and how they metabolize xenobiotics.

Neutrophils as a specific target for melatonin and kynuramines: effects on cytokine release

A growing body of evidence suggests that the pineal hormone, melatonin, has immunomodulatory properties, although very little is known about its effect on leukocytes. Therefore, we aimed to investigate the effect of melatonin and its oxidation product N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) on cytokine production by neutrophils and peripheral blood mononuclear cells (PBMCs). AFMK (0.001-1 mM) inhibits the lipopolysaccharide (LPS)-mediated production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) more efficiently in neutrophils than PBMCs. Moreover, the inhibitory activity of AFMK is stronger than that of melatonin. Interestingly, monocytes efficiently oxidize melatonin to AFMK. We conclude that neutrophils are one of the main targets for melatonin and that at least part of the effects described for melatonin on immune cells may be due to its oxidation product, AFMK. We also consider that the oxidation of melatonin may be an important event in the cross-talking between neutrophils and monocytes.

mRNA expression and release of interleukin-8 induced by serum amyloid A in neutrophils and monocytes

The acute phase response is a systemic reaction to inflammatory processes characterized by multiple physiological adaptations, including the hepatic synthesis of acute-phase proteins. In humans, serum amyloid A (SAA) is one of the most prominent of these proteins. Despite the huge increase of serum levels of SAA in inflammation, its biological role remains to be elucidated, even though SAA is undoubtedly active in neutrophils. In a previous study, we reported that SAA induces the release of tumor necrosis factor-alpha, interleukin (IL)-1beta and IL-8 from human blood neutrophils. Here, we extend our earlier study, focusing on the effect of SAA on neutrophil IL-8 transcription and on the signaling pathways involved. We demonstrate herein that SAA, in relatively low concentrations (0.4-100 microg/ml) compared with those found in plasma in inflammatory conditions, induces a dose-dependent release of IL-8 from neutrophils. The p38 mitogen-activated protein kinase inhibitor SB 203580 inhibits the IL-8 mRNA expression and the release of protein from neutrophils. The release of IL-8 from SAA-stimulated neutrophils is strongly suppressed by the addition of N-acetyl-l-cysteine, alpha-mercaptoethanol, glutathione, and dexamethasone. SAA also induces IL-8 expression and release from monocytes. In conclusion, SAA appears to be an important mediator of the inflammatory process, possibly contributing to the pool of IL-8 produced in chronic diseases, which may play a role in degenerative diseases.

High concentrations of the melatonin metabolite, N1-acetyl-N 2-formyl-5-methoxykynuramine, in cerebrospinal fluid of patients with meningitis: a possible immunomodulatory mechanism

Abstract:  We evaluated the presence of the melatonin metabolite N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (AFMK), in cerebrospinal fluid (CSF) of patients with viral meningitis (n = 20) and control samples (n = 8) and correlate AFMK levels with inflammatory markers such as cellularity, protein, tumor necrosis factor (TNF)‐α, interleukin (IL)‐8 and IL‐1β levels. A portion of the CSF was extracted with dichloromethane (1:5) and analyzed by high‐performance liquid chromatography (HPLC) under standardized conditions for AFMK. AFMK was detected in 16 of 20 CSF samples of patients with viral meningitis; the concentration of AFMK was found to be above the quantification limit (50 nmol/L) in six of these samples. AFMK was not detected in any of the eight control samples. The samples were classified into groups according to AFMK levels: undetectable (<10 nmol/L, group I), detectable but below the quantification limit (< 50 nmol/L, group II), and quantified (>50 nmol/L, group III). Group II presented the highest levels of proteins and IL‐8, whereas group III showed the lowest levels of the inflammatory parameters. This study supports our hypothesis that inflammation favors the formation of AFMK and that this compound has immunomodulatory activity in vivo.

Oxidation of melatonin and its catabolites, N-1-acetyl-N (2)-formyl-5-methoxykynuramine and N-1-acetyl-5-methoxykynuramine, by activated leukocytes

Abstract:  N 1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (AFMK) and N1‐acetyl‐5‐methoxykynuramine (AMK), two melatonin catabolites, have been described as potent antioxidants. We aimed to follow the kinetics of AFMK and AMK formation when melatonin is oxidized by phorbol myristate acetate (PMA) and lipopolysaccharide (LPS)‐activated leukocytes. An HPLC‐based method was used for AFMK and AMK determination in neutrophil and peripheral blood mononuclear cell cultures supernatants. Samples were separated isocratically on a C18 reverse‐phase column using acetonitrile/H2O (25:75) as the mobile phase. AFMK was detected by fluorescence (excitation 340 nm and emission 460 nm) and AMK by UV‐VIS absorbance (254 nm). Activation of neutrophils and mononuclear cells with PMA produces larger amounts of AFMK than activation with LPS, probably due to the lower levels of reactive oxygen species formation and myeloperoxidase (MPO) degranulation that occurs when cells are stimulated with LPS. The concentration of AMK found in the supernatant was about 5–10% (from 18‐hr cultures) compared with AFMK. This result may reflect its reactivity. Indeed AMK, but not AFMK, is easily oxidized by activated neutrophils in a MPO and hydrogen peroxide‐dependent reaction. In conclusion, we defined a simple procedure for the determination of AFMK and AMK in biological samples and demonstrated the capacity of leukocytes to oxidize melatonin and AMK.

The acute phase protein serum amyloid A primes neutrophils

We studied here the effect of the acute phase protein serum amyloid A (SAA) on the oxidative burst of neutrophils. Incubation of neutrophils with SAA increased the rate of oxygen uptake and the production of reactive oxygen species of neutrophils activated with opsonized zymosan (OZ). The increment in the neutrophil oxidative burst was dependent on SAA concentration in the range of 3-33 microg protein ml(-1) and was observed only in the presence of a relatively low amount of OZ (1 x 10(6) particles ml(-1)). SAA did not affect oxygen consumption and reactive oxygen production triggered by other stimuli, such as f-Met-Leu-Phe, phorbol myristate acetate or non-opsonized zymosan. Our finding points to a priming effect of SAA probably associated with mobilization of receptors for opsonized particles and strengthens the role of SAA as an effector of neutrophil functions in inflammation.

Chlorophyll: an efficient detector of electronically excited species in biochemical systems

Micelle-solubilized chlorophyll efficiently detects electronically excited species generated in enzymatic systems. In most, if not all, systems the chemiexcited species is formed in the triplet state; chlorophyll fluorescence is observed as result of energy transfer. Red emission can also be elicited from chlorophyll in chloroplasts or bound to microsomes.