International Immunopharmacology 11 (2011) 194–198
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Effect of the lipopolysaccharide antagonist Planktothrix sp. FP1 cyanobacterial extract on human polymorphonuclear leukocytes Ramòna Consuèlo Maio a, Marco Cosentino a, Carlo Rossetti b, Monica Molteni b, Sergio Lecchini a, Franca Marino a,⁎ a b
Department of Clinical Medicine, Section of Experimental and Clinical Pharmacology, University of Insubria, Varese, Italy Department of Structural and Functional Biology, University of Insubria, Varese, Italy
a r t i c l e
i n f o
Article history: Received 11 June 2010 Received in revised form 6 October 2010 Accepted 6 November 2010 Available online 27 November 2010 Keywords: Cyanobacterial LPS antagonist Human polymorphonuclear leukocytes Reactive oxygen species Interleukin-8 Tumor necrosis factor-alpha Lipopolysaccharide
a b s t r a c t CyP is a lipopolysaccharide (LPS)-like molecule extracted from the freshwater cyanobacterium Oscillatoria planktothrix FP1, which has been reported to be a potent competitive inhibitor of bacterial LPS. In the present study the ability of CyP to affect human polymorphonuclear leukocyte (PMN) function was investigated. PMNs were isolated from venous blood by standard density-gradient centrifugation. Cell migration was measured by use of the Boyden chamber assay. Interleukin (IL)-8 and tumor necrosis factor (TNF)-α production was measured using a sandwich-type enzyme-linked immunosorbent assay. PMN intracellular reactive oxygen species (ROS) levels were assessed by the use of a fluorescent probe coupled to spectrophotometry. CyP 10–100 μg/ml was chemotactic for PMNs without affecting the chemotactic response to either E. coli LPS or N-formyl-Met-Leu-Phe (fMLP). CyP per se did not affect PMN production of either IL-8 or TNF-α, but concentration-dependently reduced LPS-induced production of both cytokines. On the contrary, CyP had no effect either on fMLP-induced production of IL-8 or on PMN oxidative burst (at rest and after stimulation with fMLP), a response which is known to be independent from LPS-operated pathways. In human PMNs CyP behaves as a selective and effective LPS antagonist. These findings support the therapeutic potential of CyP in endotoxin-dependent disease. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Cyanobacteria, also termed blue-green algae, are a large group of photosynthetic oxygenic procaryotes with a high degree of biological adaptation, which represent one of the oldest forms of life on earth [1], as well as a rich source of bioactive metabolites endowed with attractive pharmacological activities, including: neuroprotective, cytotoxic, antibacterial, antifungal, antiviral, and antiinflammatory [2–7]. Lipopolysaccharides (LPS) derived from cyanobacteria differ in both chemical and biological properties from Gram-negative bacteriaderived LPS. In general, cyanobacterial LPS lack L-glycero-D-mannoheptose and phosphate groups, have long-chain saturated and unsaturated fatty acids, and very low content of 2-keto-3-deoxyoctonate [8–12]. In addition, cyanobacterial LPS exhibit lower biological activity and therefore usually result in lower – if any – toxicity, at least in rodent models [9,10]. Some of us recently extracted from the freshwater cyanobacterium O. planktothrix FP1 an LPS-like glycolipid, named CyP, which shows
⁎ Corresponding author. Department of Clinical Medicine, Section of Experimental and Clinical Pharmacology, University of Insubria, Via Ottorino Rossi n. 9, 21100 Varese VA, Italy. Tel.: + 39 0332 217410 397410, + 39 0332 217401 397401; fax: + 39 0332 217409/397409. E-mail address:
[email protected] (F. Marino). 1567-5769/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2010.11.017
very low levels of 2-keto-deoxy-octulosonic acids as well as minimal endotoxin activity [13]. In previous studies in human dendritic cells (DCs), CyP was shown to be a potent antagonist of bacterial LPS. In particular, in binding experiments CyP was shown to interact directly with the cell surface co-receptor molecule MD-2 (CD14) and to efficiently compete with LPS for binding to the Toll-like receptor 4 (TLR4)–MD-2 receptor complex [13]. Depending on the time of addition, CyP could either completely block LPS-induced activation of the cells or just prevent the secretion of cytokines without affecting phenotypic maturation, while in in vivo experiments CyP effectively protected mice from lethal LPS-induced shock [13] and inhibited the secretion of the proinflammatory cytokines in a human whole-blood model of meningococcal septicaemia [14], as well as the Escherichia coli LPS-induced inflammatory response in porcine whole blood [15]. Polymorphonuclear leukocytes (PMNs) play a critical role in innate immunity against microbial agents and are a central component of the inflammatory response. PMNs migrate to peripheral tissues where they kill pathogens through both oxidative and nonoxidative mechanisms. Oxidative mechanisms involve the production of reactive oxygen species (ROS), which can be microbicidal [16]. Non-oxidative mechanisms include phagocytosis and release of proinflammatory and microbicidal peptides and proteins [17]. PMNs produce a variety of chemokines and cytokines, such as interleukin (IL)-8 and tumor necrosis factor (TNF)-α, which act as autocrine/
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paracrine mediators, resulting in activation and recruitment of immune cells to inflammatory sites [18–20]. PMNs play also a key role in the pathogenesis of severe sepsis, which is associated with impaired migration and excessive/inappropriate activation of these cells [21,22]. Functional dysregulation of PMNs is part of the overwhelming activation of the host pathogen-recognition system (in particular of TLR4) which initiates an excessive pro-inflammatory response in the early phase of sepsis, finally leading to a severe imbalance of various body systems as a result of extensive tissue damage and/or severe infection (reviewed in Ref. [23]). The TLR4–MD-2 receptor complex is indeed expressed by PMNs and binds bacterial LPS, finally resulting in extensive activation and dysregulation of cell functions [24]. LPS-induced activation of PMNs includes enhanced migration and cytokine/chemokine production [25], but has usually no effect on the oxidative metabolism, which on the contrary is effectively activated by other microbial agents, such as N-formyl-Met-Leu-Phe (fMLP) [26]. Since CyP has been reported to be a potent competitive inhibitor of LPS at the TLR4–MD-2 receptor complex both in vitro and in vivo [13– 15], the aim of the present study was to investigate the ability of CyP to affect human PMNs and to interfere with the functional responses of these cells to LPS or fMLP. The possible influence of CyP on cytoplasmic free Ca2+ concentration ([Ca2+]i) changes was also examined. 2. Materials and methods 2.1. Cyanobacterial LPS-like product (CyP) CyP is an LPS-like glycolipid extracted from the freshwater cyanobacterium O. planktothrix FP1, as described previously [13]. Briefly, cultures from cyanobacterium O. planktothrix FP1 were grown until stationary phase in BG11 medium (Sigma-Aldrich, Milan, Italy) at 27 °C under constant cool white light irradiance (intensity: 20 μmol × m− 2 × s− 1). Extraction of LPS fraction was then performed according to Yi and Hackett [27] and purified extracts (named CyP) were finally dissolved in PBS at 2 mg/ml. Contamination of CyP was minimal and similar to that observed for commercially available gram-negative LPS products. In particular, protein contamination was b3% (wt/wt), and mean nucleic acid contamination was b30% (and consisted mainly of degraded RNA). The level of 2-keto-deoxyoctulosonic acids was low (0.15%, wt/wt), and endotoxin activity, as quantified by the Limulus amoebocyte lysate assay, was 4 endotoxin units (EU)/mg; that for E. coli LPS was 15,000 EU/mg [13]. 2.2. Cell preparation PMNs were isolated from venous blood obtained from healthy volunteers using heparinized tubes. Whole blood was allowed to sediment on dextran at 37 °C for 30 min. Supernatant was recovered and PMNs were isolated by Ficoll-Paque Plus density-gradient centrifugation as described [28]. Contaminating erythrocytes were eliminated by 10 min hypotonic lysis in distilled water with added (g/l): NH4Cl 8.25, KHCO3 1.00, EDTA 0.04. Cells were then washed three times in NaCl 0.15 M and resuspended in 1 ml Ca2+/Mg2+-free PBS (composition as follows [g/l]: NaHPO4 · H2O 17.80, Na2HPO4 · H2O 13.80, NaCl 8.80) with added BSA 0.25%. Purity and viability of PMN preparations were always N95% and no platelets or erythrocytes could be detected by either light microscopic examination or flow cytometric analysis. 2.3. Cell migration Cell migration was measured by use of the Boyden chamber assay [29]. PMNs were resuspended at 1 × 106/ml in RPMI 1640 with 0.5% BSA before being placed in the top well of the Boyden chamber. The two compartments of the chamber were separated by a 3 μm pore
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cellulose nitrate filter (Millipore Corporation, Bedford, MA, USA). After an incubation period of 1.5 h at 37 °C, the filter was recovered, dehydrated, fixed, and finally stained with hematoxilin/eosin. Migration into the filter was quantified by microscopically measuring the distance (in μm) from the surface of the filter to the leading front of cells. 2.4. IL-8 and TNF-α assay PMNs were resuspended at the concentration of 1 × 107 cells/ml in RPMI and incubated at 37 °C for 5 h. The effect of CyP on IL-8 and TNFα production was assessed on cells at rest and treated with 0.1 μM Nformyl-Met-Leu-Phe (fMLP; Sigma-Aldrich) or with 1 μg/ml E. coli LPS (Sigma-Aldrich). CyP was added to the cells at the beginning of the incubation period. When cells were exposed to fMLP or LPS, these agents were added 1 h after CyP. After incubation, the cells were centrifuged (600 g, 5 min, 20 °C) and the supernatant was recovered for IL-8 and TNF-α assay. Cytokine levels in PMN supernatants were quantified using a sandwich-type enzyme-linked immunosorbent assay (ELISA kit; Amersham Biosciences, Little Chalfont, UK). The detection limit of the assay was 1 pg/ml. 2.5. Reactive oxygen species generation Intracellular ROS levels were assessed by using the redox-sensitive dye dichlorodihydrofluorescein-diacetate (C-DCDHF-DA; Molecular Probe, Eugene, OR, USA) as described previously [30]. ROS levels were detected with a Perkin-Elmer LS-50B spectrofluorimeter using an excitation wavelength of 488 nm; fluorescence emission was collected at 525 nm. Data were then expressed as arbitrary units of fluorescence intensity (FI). The effect of CyP on ROS generation was tested on resting cells and on cells treated with 0.1 μM fMLP or with 1 μg/ml LPS. In each experiment, CyP was added to the cells after a 60s resting period, alone or followed (60 s after) by fMLP or LPS, and subsequently ROS changes were calculated as the area under the ROS levels-vs-time curve (AUC), over a 30-min period. The response of the spectrofluorimeter was linearly related to ROS concentrations in the 0.300–0.900 FI range. 2.6. Measurement of [Ca2+]i [Ca2+]i measurement in human PMNs was performed as previously described [31], by using a spectrofluorimetric method according to Grynkiewicz et al. [32] with modifications. [Ca2+]i changes were calculated as the difference (Δ) between the highest values (peak levels) reached after the addition of a given agent and the mean 1 min pretreatment values (resting levels) in each experiment. 2.7. Statistical analysis Results are presented as means ± SEM. The statistical significance of the differences was determined by Student's t test or by one-way analysis of variance followed by Dunnett or Bonferroni post test, as appropriate, using a commercial software (Prism 2.0, GraphPad Software, San Diego, CA, USA). 3. Results 3.1. Migration Spontaneous migration of PMNs was 27.0±6.9 μm (n =5), and was increased in the presence of CyP 10–100 μg/ml in a concentrationdependent manner. Checkerboard analysis showed that the effect of CyP was chemotactic and not merely chemokinetic (Table 1). CyP (100 μg/ml) did not affect the chemotactic response to either LPS 1 μg/ml (56.9±17.0 μm with LPS alone vs 49.2±11.1 μm with LPS+CyP, n =5;
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Table 1 Checkerboard analysis of the effect of CyP on PMN migration. Upper compartment [CyP] (μg/ml) Lower compartment
0 10 100
0
10
100
1.00 ± 0.11 1.15 ± 0.35 0.92 ± 0.13
1.56 ± 0.45⁎
1.91 ± 0.33# 1.02 ± 0.51 1.20 ± 0.47
1.12 ± 0.47 1.25 ± 0.55
Results are expressed as chemotactic index (C.I.), i.e. the ratio between the distance of cells that migrated in the presence and in the absence of the test substance(s), and are reported as means ± SEM of 3–6 separate experiments. ⁎ = P b 0.05. # = P b 0.01 vs control.
PN 0.05) or fMLP 0.1 μM (66.4±19.9 μm with fMLP alone vs 47.5± 6.8 μm with fMLP+CyP, n=5; PN 0.05). 3.2. IL-8 and TNF-α production Spontaneous release of IL-8 after 5 h incubation by resting PMNs was 110.9 ± 34.4 pg/ml (n = 3–5). LPS 1 μg/ml increased IL-8 production, and CyP concentration-dependently reduced its effect without affecting resting levels of this cytokines (Fig. 1). We have also tested the effect of CyP on IL-8 production at different times of incubation. When PMNs were cultured in the presence of Cyp (10 μg/ml) for 1 or 8 h, no differences was observed in IL-8 production with respect to control values (Table 2). In addition, in the same culture conditions, LPS-induced IL-8 production was not reverted by the presence of Cyp (10 μg/ml) (P N 0.05 vs LPS alone in both cases, Table 2). Spontaneous release of TNF-α by resting PMNs at 5 h of incubation was 4.0 ± 2.5 pg/ml (n = 3–5), LPS (1 μg/ml) increased TNF-α production and CyP concentration-dependently reduced the TNF-α levels without affecting resting levels (Fig. 2). Incubation of PMNs with fMLP 0.1 μM increased the levels of IL-8 (up to 428.3± 90.9 pg/ml, n =3; P b 0.05 vs resting levels) but not of TNF-α (7.0 ±4.5 pg/ml, n =3; P N 0.05 vs resting levels), and the presence of CyP 10 μg/ml failed to modify such effects (IL-8= 373.08 ±53.70 pg/ml and TNF-α= 7.1± 5.7 pg/ml, both n = 3; P N 0.05 vs fMLP alone). 3.3. ROS generation Resting levels of ROS were 869.1 ± 375.0 (n = 3). As expected, fMLP 0.1 μM significantly increased ROS generation up to 2487.3 ± 455.8
Fig. 1. Effect of CyP on IL-8 production in human PMNs cultured for 5 h. Effect of CyP (1– 10 μg/ml) on resting cells (empty columns) and stimulated with LPS 1 μg/ml (filled columns). Each column is the means ± SEM of 4–10 separate experiments. * = P b 0.05 and ** = P b 0.01 vs control (CyP = 0); # = P b 0.05 vs LPS.
(n= 3; P b 0.05 vs resting levels), while LPS 1 μg/ml had no effect (1212.3 ± 244.3, n = 3; P N 0.05 vs resting levels). CyP (100 μg/ml) did not modify to any significant extent ROS levels, either in resting conditions (831.2 ± 299.0, n = 3; P N 0.05 vs resting levels) or in the presence of fMLP (2045.9 ± 366.0, n = 3; P N 0.05 vs fMLP alone) or LPS (1169.2 ± 202.8, n = 3; P N 0.05 vs LPS alone). 3.4. [Ca2+]i Resting [Ca2+]i levels were 83.4 ± 16.2 nM (n = 3). Incubation with fMLP 0.1 μM induced a rapid and transient rise of [Ca2+]i up to 268.2 ± 22.0 nM (n = 3; P b 0.001 vs resting levels). CyP (10 μg/ml) per se had no effect either on [Ca2+]i (66.8 ± 22.6 nM, n = 3; P N 0.05 vs resting levels) or on fMLP-induced [Ca2+]i rise (231.4 ± 5.5 nM, n = 3; P N 0.05 vs fMLP alone). 4. Discussion In the present study, we characterized the effects of the LPS antagonist Planktothrix sp. FP1 cyanobacterial extract (CyP) on human PMNs. Results show that CyP selectively reduces the stimulatory effects of LPS on IL-8 and TNF-α production, but not on cell migration, with no effect on the functional responses of these cells to fMLP. CyP per se increased PMN migration, with no effect on IL-8 or TNF-α production nor on the oxidative metabolism or the [Ca2+]i levels of these cells. CyP-induced inhibition of LPS-stimulated IL-8 and TNF-α production in human PMNs is in agreement with previous observations showing that CyP was able to inhibit N. meningitidis LPS-induced secretion of TNF-α, IL-1β, IL-6, IL-8 and monocyte chemoattractant protein (MCP)-1 [14], as well as E. coli LPS-induced secretion of TNFα, IL-1β and IL-8 in porcine whole blood [15], and E. coli LPS-induced production of TNF-α, IL-6, and IL-12p70 in human DCs [13]. Moreover, in the human PMNs the prolonged LPS-induced IL-8 production (8 h incubation) was not inhibited by addition of CyP; we hypothesize that at this time the effect of CyP could be masked by the considerable increase of spontaneous release of IL-8 observed with respect to values measured at 5 h. LPS binds with high affinity TLR4–MD-2 receptor complex expressed by immune cells and in particular by PMNs [33,34]. Indeed, soluble MD-2, an endotoxin binding unit, binds LPS directly with high affinity and the LPS/MD-2 complex forms a ligand that activates TLR4 positive cells [35]. In previous studies, CyP was shown to bind selectively to MD-2 and to efficiently compete with LPS for binding to the TLR4-MD-2 receptor complex and the downstream activation of NF-kB [13,14]. CyP, as antagonist of bacterial LPS, completely block the human DCs activation LPS-induced and did not induce gene transcripts of cytokines such as TNF-α, IL-6, IL-10 and chemokines. Moreover, in the human DCs, LPS-induced TNF-α and IL-6 production were inhibited in a dose-dependent fashion by addition of CyP (20 μg/ml) [13]. On the contrary, in the same model, CyP not interfere with peptidoglican, poly(I:C), R848 (which trigger TLR2, TLR3, and TLR8,
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Table 2 Effect of CyP on resting and LPS-induced IL-8 production in human PMNs at different times of incubation. IL-8 production (pg/ml)
Control LPS (1 μg/ml) CyP (10 μg/ml) CyP (10 μg/ml) + LPS (1 μg/ml)
Time 1h
8h
139.80 ± 40.72 228.14 ± 64.38 151.52 ± 69.77 215.17 ± 58.32
411.50 ± 29.44 1255.00 ± 326.78⁎⁎ 605.50 ± 304.26 1086.00 ± 203.23⁎
Results are expressed in pg/ml and are reported as means ± SEM of 4 separate experiments. ⁎ = P b 0.05. ⁎⁎ = P b 0.005 vs control.
respectively) IL-1β, or CD40L indicating that it does not interfere with TLR9 stimulation and suggesting further that CyP behaves as a selective inhibitor of the LPS-TLR4 axis. In our experiments, CyP effectively antagonized LPS but not fMLP. Indeed, CyP had no effect on several functional responses to fMLP, including oxidative burst, IL-8 production and [Ca2+]i rise. The tripeptide fMLP is the main chemotactic factor produced by E. coli [36] and exerts its effects by binding to FPRs, which are classical Gprotein-coupled receptors characterized by seven hydrophobic transmembrane segments connected by hydrophilic domains [37]. FPRs may bind also some antimicrobial peptides produced by immune cells, such cathepsin G, contributing to their physiological effects [38]. The present results thus strengthen the notion that CyP does not affect cellular pathways other than those acted upon by LPS molecules. The incubation of human PMNs with CyP alone had no effect on either IL-8 or TNF-α production, as well as on the oxidative burst or on [Ca2+]i levels. In these cells however CyP had a clear chemoattractant effect which was similar to that exerted by either LPS or fMLP. It was previously shown that in human whole blood CyP at high concentrations exerted a stimulatory effect on IL-8 and MCP-1 production [15]. In the present study we observed no stimulatory effect on IL8 production by PMNs, however the chemotactic effect displayed by CyP on these cells may suggest that this compound is endowed with some degree of intrinsic activity. It was previously shown however that the endotoxin activity of CyP was only 4 EU/mg in comparison to that for E. coli LPS which was 15,000 EU/mg [13]. Alternatively, it cannot be excluded that the chemotactic response to CyP depends upon the activation of TLR4-MD-2-independent pathways, although the lack of additive/synergic effect with LPS or fMLP does not support this possibility.
Fig. 2. Effect of CyP TNF-α production in human PMNs cultured for 5 h. Effect of CyP (1–10 μg/ml) on in resting cells (empty columns) and in cells stimulated with LPS 1 μg/ml (filled columns). Each column is the means ± SEM of 4–10 separate experiments. * = P b 0.05 and ** = P b 0.01 vs control (CyP = 0); # = P b 0.05 and ## = P b 0.01 vs LPS.
5. Conclusions In summary, we showed that CyP behaves as a selective and effective LPS antagonist even in human PMNs. In view of the role of these cells in endotoxin-dependent disease [22,23], these findings further support the therapeutic potential of CyP. In particular, the chemotactic effect of CyP deserves further consideration, also in view of the specific impairment of PMN migration which is a hallmark of severe sepsis [21]: future studies should include the effect of CyP on PMNs from patients, to assess whether this molecule is able to revert their functional impairment. In any case, available evidence now strongly warrants careful clinical evaluation of the therapeutic potential of CyP. Authors' contributions RCM and FM performed the experiments and drafted the manuscript together with MC; CR and MM isolated and characterized the extract and contributed to define the experimental design and procedures; MC and FM conceived the study and performed the analysis of the data; SL participated to data analysis and interpretation. All authors read and approved the final manuscript. References [1] Stanier RY, Cohen-Bazire G. Phototrophic prokaryotes: the cyanobacteria. Annu Rev Microbiol 1977;31:225–74. [2] Romay C, Ledon N, Gonzalez R. Further studies on anti-inflammatory activity of phycocyanin in some animal models of inflammation. Inflamm Res 1998;47: 334–8. [3] Oufdou K, Mezrioui N, Oudra B, Loudiki M, Barakate M, Sbiyyaa B. Bioactive compounds from Pseudanabaena species (Cyanobacteria). Microbios 2001;106: 21–9. [4] Shi Q, Cui J, Zhang J, Kong FX, Hua ZC, Shen PP. Expression modulation of multiple cytokines in vivo by cyanobacteria blooms extract from Taihu Lake, China. Toxicon 2004;44:871–9. [5] Zainuddin EN, Mundt S, Wegner U, Mentel R. Cyanobacteria a potential source of antiviral substances against influenza virus. Med Microbiol Immunol 2002;191: 181–2. [6] Garbacki N, Gloaguen V, Damas J, Hoffmann L, Tits M, Angenot L. Inhibition of croton oil-induced oedema in mice ear skin by capsular polysaccharides from cyanobacteria. Naunyn Schmied Arch Pharmacol 2000;361:460–4. [7] Prinsep MR, Thomson RA, West ML, Wylie BL. Tolypodiol, an antiinflammatory diterpenoid from the cyanobacterium Tolypothrix nodosa. J Nat Prod 1996;59: 786–8. [8] Buttke TM, Ingram LO. Comparison of lipopolysaccharides from Agmenellum quadruplicatum to Escherichia coli and Salmonella typhimurium by using thin-layer chromatography. J Bacteriol 1975;124:1566–73. [9] Keleti G, Sykora JL. Production and properties of cyanobacterial endotoxins. Appl Environ Microbiol 1982;43:104–9. [10] Keleti G, Sykora JL, Lippy EC, Shapiro MA. Composition and biological properties of lipopolysaccharides isolated from Schizothrix calcicola (Ag.) Gomont (Cyanobacteria). Appl Environ Microbiol 1979;38:471–7. [11] Mikheyskaya LV, Ovodova RG, Ovodov YS. Isolation and characterization of lipopolysaccharides from cell walls of blue-green algae of the genus. Phormidium J Bacteriol 1977;130:1–3. [12] Weckesser J, Katz A, Drews G, Mayer H, Fromme I. Lipopolysaccharide containing l-acofriose in the filamentous blue-green alga Anabaena variabilis. J Bacteriol 1974;120:672–8. [13] Macagno A, Molteni M, Rinaldi A, Bertoni F, Lanzavecchia A, Rossetti C, et al. A cyanobacterial LPS antagonist prevents endotoxin shock and blocks sustained TLR4 stimulation required for cytokine expression. J Exp Med 2006;203:1481–92.
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