Inhibitory activity of liposomal flavonoids during oxidative metabolism of human neutrophils upon stimulation with immune complexes and phorbol ester

Context and objective: The massive production of reactive oxygen species by neutrophils during inflammation may cause damage to tissues. Flavonoids act as antioxidants and have anti-inflammatory effects. In this study, liposomes loaded with these compounds were evaluated as potential antioxidant carriers, in attempt to overcome their poor solubility and stability. Materials and methods: Liposomes containing quercetin, myricetin, kaempferol or galangin were prepared by the ethanol injection method and analyzed as inhibitors of immune complex (IC) and phorbol ester-stimulated neutrophil oxidative metabolism by luminol (CLlum) and lucigenin-enhanced (CLluc) chemiluminescence (CL) assays. The mechanisms involved this activity of liposomal flavonoids, such as cytotoxicity and superoxide anion scavenging capacity, and their effect on phagocytosis of ICs were also investigated. Results and discussion: The results showed that the inhibitory effect of liposomal flavonoids on CLlum and CLluc is inversely related to the number of hydroxyl groups in the flavonoid B ring. Moreover, phagocytosis of liposomes by neutrophils does not seem to necessarily promote such activity, as the liposomal flavonoids are also able to reduce CL when the cells are pretreated with cytochalasin B. Under assessed conditions, the antioxidant liposomes are not toxic to the human neutrophils and do not interfere with IC-induced phagocytosis. Conclusion: The studied liposomes can be suitable carriers of flavonoids and be an alternative for the treatment of diseases in which a massive oxidative metabolism of neutrophils is involved.

Human Leucocytes Response to Viable, Extended Freeze-Drying or Heat-Killed Mycobacterium bovis bacillus Calmette-Guerin

We investigated the effects of viable, extended freeze-drying (EFD) or heat-killed (HK) Mycobacterium bovis bacillus CalmetteGuerin (BCG) in respiratory burst activity, gene expression of CYBB and NCF1 encoding components of the human phagocyte nicotinamide adenine dinucleotide (NADPH) oxidase, TLR2 expression, and in IL-10 and TNF-a cytokine production by human peripheral blood mononuclear cells (PBMCs). Viable BCG significantly inhibited TLR2 and CYBB gene expression, as well as superoxide release by human PBMC. All BCG stimuli augmented IL-10 release, but only HK BCG or viable BCG increased TNF-a release by PBMCs. Our studies show that viable BCG can impair the NADPH oxidase system activation and the TLR2 route in human PBMCs. As well, different BCG preparations can distinctly influence cytokine production by human PBMCs.

BAY 41-2272, a soluble guanylate cyclase agonist, activates human mononuclear phagocytes

BACKGROUND AND PURPOSE Phagocyte function is critical for host defense against infections. Defects in phagocytic function lead to several primary immunodeficiencies characterized by early onset of recurrent and severe infections. In this work, we further investigated the effects of BAY 41-2272, a soluble guanylate cyclase (sGC) agonist, on the activation of human peripheral blood monocytes (PBM) and THP-1 cells. EXPERIMENTAL APPROACH THP-1 cells and PBM viability was evaluated by methylthiazoletetrazolium assay; reactive oxygen species production by lucigenin chemiluminescence; gene and protein expression of NAPDH oxidase components by qRT-PCR and Western blot analysis, respectively; phagocytosis and microbicidal activity by co-incubation, respectively, with zymosan and Escherichia coli; and cytokine release by elisa. KEY RESULTS BAY 41-2272, compared with the untreated group, increased spreading of monocytes by at least 35%, superoxide production by at least 50%, and gp91PHOX and p67PHOX gene expression 20 to 40 times, in both PBM and THP-1 cells. BAY 41-2272 also augmented phagocytosis of zymosan particles threefold compared with control, doubled microbicidal activity against E. coli and enhanced the release of TNF-a and IL-12p70 by both PBM and THP-1 cells. Finally, by inhibiting sGC with ODQ, we showed that BAY 41-2272-induced superoxide production and phagocytosis is not dependent exclusively on sGC activation. CONCLUSIONS AND IMPLICATIONS In addition to its ability to induce vasorelaxation and its potential application for therapy of vascular diseases, BAY 41-2272 was shown to activate human mononuclear phagocytes. Hence, it is a novel pro-inflammatory drug that may be useful for controlling infections in the immunocompromised host.

Activation profile of CXCL8-stimulated neutrophils and aging

Neutrophils are pivotal effector cells of innate immunity representing the first line of defense against aggression. They are the first cells to arrive at the site of the aggression, where they can directly eliminate the invading microorganisms. Their activation and recruitment into peripheral tissues is indispensable for host defense. With aging, there are alterations of the receptor by driven functions of human neutrophils as a decrease in the functional changes in signaling elicited by specific receptors, as CXCR1. We investigated the activation of neutrophils from elderly after the cells were cultivated with CXCL8. Although, CXCL8 induced elastase (ELA) secretion, data showed neither myeloperoxidase (MPO) activity nor production of IL-6, IL-10, GM-CSF by neutrophils from elderly compared with young individuals. On the other hand, in the presence of only LPS or LPS associated with CXCL8 neutrophils from elderly individuals, there were significant levels of IL-6, IL-10, GM-CSF but not MPO. These results indicate that neutrophils from elderly do not respond to CXCL8 stimulus, but they are activated by LPS to produce cytokines. However, MPO activity from elderly individuals was not different in the presence or absence of LPS and CXCL8

M-ficolin and leukosialin (CD43): new partners in neutrophil adhesion

M-ficolin specificity for sialylated ligands prompted us to investigate its interactions with the main membrane sialoprotein of human neutrophils, CD43. rM-ficolin bound CD43 and prevented the access of anti-CD43 mAb. Moreover, rM-ficolin reacted exclusively with CD43 on Western blots of neutrophil lysate. We confirmed that M-ficolin is secreted by fMLP-activated neutrophils, and this endogenous M-ficolin also binds to CD43 and competes with anti-CD43 mAb. Anti-CD43 antibody cross-linking or fMLP resulted in M-ficolin and CD43 colocalization on polarized neutrophils. The binding of rM-ficolin to resting neutrophils induced cell polarization, adhesion, and homotypic aggregation as anti-CD43 mAb. The M-ficolin Y271F mutant, unable to bind sialic acid, neither reacted with neutrophils nor modulated their functions. Finally, rM-ficolin activated the lectin complement pathway on neutrophils. These results emphasize a new function of M-ficolin, different from ficolin pathogen recognition, i.e., a participation to neutrophil adhesion potentially important in early inflammation, as nanomolar agonist concentrations are sufficient to mobilize M-ficolin to the neutrophil surface. This multivalent lectin could then endow the antiadhesive CD43, essentially designed to prevent leukocyte aggregation in the blood flow, with new adhesive properties and explain, at least in part, dual-adhesive/antiadhesive roles of CD43 in neutrophil recruitment. J. Leukoc. Biol. 91: 469-474; 2012.

GM-CSF Priming Drives Bone Marrow-Derived Macrophages to a Pro-Inflammatory Pattern and Downmodulates PGE(2) in Response to TLR2 Ligands

In response to pathogen recognition by Toll-like receptors (TLRs) on their cell surface, macrophages release lipid mediators and cytokines that are widely distributed throughout the body and play essential roles in host responses. Granulocyte macrophage colony-stimulating factor (GM-CSF) is important for the immune response during infections to improve the clearance of microorganisms. In this study, we examined the release of mediators in response to TLR2 ligands by bone marrow-derived macrophages (BMDMs) primed with GM-CSF. We demonstrated that when stimulated with TLR2 ligands, non-primed BMDMs preferentially produced PGE(2) in greater amounts than LTB4. However, GM-CSF priming shifted the release of lipid mediators by BMDMs, resulting in a significant decrease of PGE(2) production in response to the same stimuli. The decrease of PGE(2) production from primed BMDMs was accompanied by a decrease in PGE-synthase mRNA expression and an increase in TNF-alpha and nitric oxide (NO) production. Moreover, some GM-CSF effects were potentiated by the addition of IFN-gamma. Using a variety of TLR2 ligands, we established that PGE(2) release by GM-CSF-primed BMDMs was dependent on TLR2 co-receptors (TLR1, TLR6), CD14, MyD88 and the nuclear translocation of NF kappa B but was not dependent on peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activation. Indeed, GM-CSF priming enhanced TLR2, TLR4 and MyD88 mRNA expression and phospho-I kappa B alpha formation. These findings demonstrate that GM-CSF drives BMDMs to present a profile relevant to the host during infections.

WBC count and functional changes induced by co-administration of clofazimine and clarithromycin, in single and multiple doses, in Wistar rats

Clofazimine and clarithromycin are used to treat leprosy and infections caused by Mycobacterium avium complex. Little data on the toxicity of co-administration of these two drugs are available. Here we evaluated the potential adverse effects of polytherapy with these two drugs in male Wistar rats by determining WBCs counts and other blood cell counts, neutrophilic phagocytosis, and burst oxidative, by flow cytometry. We observed an increase in WBCs, in multiple-dose regimens, and in polymorphonuclear cells, in both single- clarithromycin only and multiple dose regimens. We also observed a reduction in mononuclear cell counts in single and multiple doses. The drugs seem to reverse the mononuclear and polymorphonuclear cell ratio. An increase in oxidative burst was observed in animals treated with the drugs administered either individually or combined. In conclusion, clofazimine and clarithromycin change WBCs counts. Our results may contribute for a better understanding of the mechanisms related to the effects of co-administrating the two drugs.

5-Lipoxygenase Deficiency Impairs Innate and Adaptive Immune Responses during Fungal Infection

5-lipoxygenase-derived products have been implicated in both the inhibition and promotion of chronic infection. Here, we sought to investigate the roles of endogenous 5-lipoxygenase products and exogenous leukotrienes during Histoplasma capsulatum infection in vivo and in vitro. 5-LO deficiency led to increased lung CFU, decreased nitric oxide production and a deficient primary immune response during active fungal infection. Moreover, H. capsulatum-infected 5-LO-/- mice showed an intense influx of neutrophils and an impaired ability to generate and recruit effector T cells to the lung. The fungal susceptibility of 5-LO-/- mice correlated with a lower rate of macrophage ingestion of IgG-H. capsulatum relative to WT macrophages. Conversely, exogenous LTB4 and LTC4 restored macrophage phagocytosis in 5-LO deficient mice. Our results demonstrate that leukotrienes are required to control chronic fungal infection by amplifying both the innate and adaptive immune response during histoplasmosis.

Signaling Events During Cyclic Guanosine Monophosphate-Regulated Pigment Aggregation in Freshwater Shrimp Chromatophores

Crustacean color change results partly from granule aggregation induced by red pigment concentrating hormone (RPCH). In shrimp chromatophores, both the cyclic GMP (3', 5'-guanosine monophosphate) and Ca2+ cascades mediate pigment aggregation. However, the signaling elements upstream and downstream from cGMP synthesis by GC-S (cytosolic guanylyl cyclase) remain obscure. We investigate post-RPCH binding events in perfused red ovarian chromatophores to disclose the steps modulating cGMP concentration, which regulates granule translocation. The inhibition of calcium/calmodulin complex (Ca2+/CaM) by N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7) induces spontaneous aggregation but inhibits RPCH-triggered aggregation, suggesting a role in pigment aggregation and dispersion. Nitric oxide synthase inhibition by N omega-nitro-L-arginine methyl ester hydrochloride (L-NAME) strongly diminishes RPCH-induced aggregation; protein kinase G inhibition (by rp-cGMPs-triethylamine) reduces RPCH-triggered aggregation and provokes spontaneous dispersion, disclosing NO/PKG participation in aggregation signaling. Myosin light chain phosphatase inhibition (by cantharidin) accelerates RPCH-triggered aggregation, whereas Rho-associated protein kinase inhibition (by Y-27632, H-11522) reduces RPCH-induced aggregation and accelerates dispersion. MLCP (myosin light chain kinase) and ROCK (Rho-associated protein kinase) may antagonistically regulate myosin light chain (MLC) dephosphorylation/phosphorylation during pigment dispersion/aggregation. We propose the following general hypothesis for the cGMP/Ca2+ cascades that regulate pigment aggregation in crustacean chromatophores: RPCH binding increases Ca2+ (int), activating the Ca2+/CaM complex, releasing NOS-produced nitric oxide, and causing GC-S to synthesize cGMP that activates PKG, which phosphorylates an MLC activation site. Myosin motor activity is initiated by phosphorylation of an MLC regulatory site by ROCK activity and terminated by MLCP-mediated dephosphorylation. Qualitative comparison reveals that this signaling pathway is conserved in vertebrate and invertebrate chromatophores alike.

Partial Replacement of omega-6 Fatty Acids With Medium-Chain Triglycerides, but Not Olive Oil, Improves Colon Cytokine Response and Damage in Experimental Colitis

Background: Soybean oil is rich in omega-6 fatty acids, which are associated with higher incidence and more severe cases of inflammatory bowel diseases. The authors evaluated whether partial replacement of soybean oil by medium-chain triglycerides (MCTs) or olive oil influenced the incidence and severity of experimental ulcerative colitis by using different parenteral lipid emulsions (LEs). Methods: Wistar rats (n = 40) were randomized to receive parenteral infusion of the following LE: 100% soybean oil (SO), 50% MCT mixed with 50% soybean oil (MCT/SO), 80% olive oil mixed with 20% soybean oil (OO/SO), or saline (CC). After 72 hours of infusion, acetic acid experimental colitis was induced. After 24 hours, colon histology and cytokine expression were analyzed. Results: SO was not significantly associated with overall tissue damage. MCT/SO was not associated with necrosis (P < .005), whereas OO/SO had higher frequencies of ulcer and necrosis (P < .005). SO was associated with increased expression of interferon-gamma (P = .005) and OO/SO with increased interleukin (IL)-6 and decreased tumor necrosis factor-alpha expression (P < .05). MCT/SO appeared to decrease IL-1 (P < .05) and increase IL-4 (P < .001) expression. Conclusions: Parenteral SO with high concentration of omega-6 fatty acids was not associated with greater tissue damage in experimental colitis. SO partial replacement with MCT/SO decreased the frequency of histological necrosis and favorably modulated cytokine expression in the colon; however, replacement with OO/SO had unfavorable effects. (JPEN J Parenter Enteral Nutr. 2012; 36: 442-448)

Doxycycline ameliorates 2K-1C hypertension-induced vascular dysfunction in rats by attenuating oxidative stress and improving nitric oxide bioavailability

Vascular dysfunction associated with two-kidney, one-clip (2K-1C) hypertension may result from both altered matrix metalloproteinase (MMP) activity and higher concentrations of reactive oxygen species (ROS). Doxycycline is considering the most potent MMP inhibitor of tetracyclines and attenuates 2K-1C hypertension-induced high blood pressure and chronic vascular remodeling. Doxycycline might also act as a ROS scavenger and this may contribute to the amelioration of some cardiovascular diseases associated with increased concentrations of ROS. We hypothesized that in addition to its MMP inhibitory effect, doxycycline attenuates oxidative stress and improves nitric oxide (NO) bioavailability in 2K-1C hypertension, thus improving hypertension-induced arterial endothelial dysfunction. Sham operated or 2K-1C hypertensive rats were treated with doxycycline 30 mg/kg/day (or vehicle). After 8 weeks of treatment, aortic rings were isolated to assess endothelium dependent vasorelaxation to A23187. Arterial and systemic levels of ROS were respectively measured using dihydroethidine (DHE) and thiobarbituric acid reactive substances (TBARS). Neutrophils-derived ROS were tested in vitro using the fluoroprobe Carboxy-H(2)DCFDA and human neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA). NO levels were assessed in rat aortic endothelial cells by confocal microscopy. Aortic MMP activity was determined by in situ zymography. Doxycycline attenuated 2K-1C hypertension (169 +/- 17.3 versus 209 +/- 10.9 mm Hg in hypertensive controls, p < 0.05) and protected against hypertension-induced reduction in endothelium-dependent vasorelaxation to A23187 (p < 0.05). Doxycycline also decreased hypertension-induced oxidative stress (p <= 0.05), higher MMP activity (p < 0.01) and improved NO levels in aortic endothelial cells (p < 0.01). Therefore, doxycycline ameliorates 2K-1C hypertension-induced endothelial dysfunction in aortas by inhibiting oxidative stress generation and improving NO bioavailability, in addition to its inhibitory effects on MMP activity. (C) 2012 Elsevier Inc. All rights reserved.

PTEN Directly Activates the Actin Depolymerization Factor Cofilin-1 During PGE(2)-Mediated Inhibition of Phagocytosis of Fungi

Macrophage ingestion of the yeast Candida albicans requires its recognition by multiple receptors and the activation of diverse signaling programs. Synthesis of the lipid mediator prostaglandin E-2 (PGE(2)) and generation of cyclic adenosine monophosphate (cAMP) also accompany this process. Here, we characterized the mechanisms underlying PGE(2)-mediated inhibition of phagocytosis and filamentous actin (F-actin) polymerization in response to ingestion of C. albicans by alveolar macrophages. PGE(2) suppressed phagocytosis and F-actin formation through the PGE(2) receptors EP2 and EP4, cAMP, and activation of types I and II protein kinase A. Dephosphorylation and activation of the actin depolymerizing factor cofilin-1 were necessary for these inhibitory effects of PGE(2). PGE(2)-dependent activation of cofilin-1 was mediated by the protein phosphatase activity of PTEN (phosphatase and tensin homolog deleted on chromosome 10), with which it directly associated. Because enhanced production of PGE(2) accompanies many immunosuppressed states, the PTEN-dependent pathway described here may contribute to impaired antifungal defenses.

Comparison of the Neutrophil Proteome in Trauma Patients and Normal Controls

Background: Neutrophils have an impressive array of microbicidal weapons, and in the presence of a pathogen, progress from a quiescent state in the bloodstream to a completely activated state. Failure to regulate this activation, for example, when the blood is flooded with cytokines after severe trauma, causes inappropriate neutrophil activation that paradoxically, is associated with tissue and organ damage. Acidic proteomic maps of quiescent human neutrophils were analyzed and compared to those of activated neutrophils from severe trauma patients. The analysis revealed 114 spots whose measured volumes differed between activated and quiescent neutrophils, with 27 upregulated and 87 downregulated in trauma conditions. Among the identified proteins, grancalcin, S100-A9 and CACNB2 reinforce observed correlations between motility and ion flux, ANXA3, SNAP, FGD1 and Zfyve19 are involved in vesicular transport and exocytosis, and GSTP1, HSPA1 HSPA1L, MAOB, UCH-L5, and PPA1 presented evidence that activated neutrophils may have diminished protection against oxidative damage and are prone to apoptosis. These are discussed, along with proteins involved in cytoskeleton reorganization, reactive oxygen species production, and ion flux. Proteins such as Zfyve19, MAOB and albumin-like protein were described for the first time in the neutrophil. In this work we achieved the identification of several proteins potentially involved in inflammatory signaling after trauma, as well as proteins described for the first time in neutrophils.

Expression of recombinant human mutant granulocyte colony stimulating factor (Nartograstim) in Escherichia coli

The human granulocyte colony stimulating factor (hG-CSF) plays an important role in hematopoietic cell proliferation/differentiation and has been widely used as a therapeutic agent for treating neutropenias. Nartograstim is a commercial G-CSF that presents amino acid changes in specific positions when compared to the wildtype form, which potentially increase its activity and stability. The aim of this work was to develop an expression system in Escherichia coli that leads to the production of large amounts of a recombinant hG-CSF (rhG-CSF) biosimilar to Nartograstim. The nucleotide sequence of hg-csf was codon-optimized for expression in E. coli. As a result, high yields of the recombinant protein were obtained with adequate purity, structural integrity and biological activity. This protein has also been successfully used for the production of specific polyclonal antibodies in mice, which could be used in the control of the expression and purification in an industrial production process of this recombinant protein. These results will allow the planning of large-scale production of this mutant version of hG-CSF (Nartograstim), as a potential new biosimilar in the market.

Human Leukocyte Death After a Preoperative Infusion of Medium/Long-Chain Triglyceride and Fish Oil Parenteral Emulsions: A Randomized Study in Gastrointestinal Cancer Patients

Background: Parenteral lipid emulsions (LEs) can influence leukocyte functions. The authors investigated the effect of 2 LEs on leukocyte death in surgical patients with gastrointestinal cancer. Material and Methods: Twenty-five patients from a randomized, double-blind clinical trial (ID: NCT01218841) were randomly included to evaluate leukocyte death after 3 days of preoperative infusion (0.2 g fat/kg/d) of an LE composed equally of medium/long-chain triglycerides and soybean oil (MCTs/LCTs) or pure fish oil (FO). Blood samples were collected before (t0) and after LE infusion (t1) and on the third postoperative day (t2). Results: After LE infusion (t1 vs t0), MCTs/LCTs did not influence cell death; FO slightly increased the proportion of necrotic lymphocytes (5%). At the postoperative period (t2 vs t0), MCTs/LCTs tripled the proportion of apoptotic lymphocytes; FO maintained the slightly increased proportion of necrotic lymphocytes (7%) and reduced the percentage of apoptotic lymphocytes by 74%. In the postoperative period, MCT/LCT emulsion increased the proportion of apoptotic neutrophils, and FO emulsion did not change any parameter of apoptosis in the neutrophil population. There were no differences in lymphocyte or neutrophil death when MCT/LCT and FO treatments were compared during either preoperative or postoperative periods. MCT/LCTs altered the expression of 12 of 108 genes related to cell death, with both pro- and antiapoptotic effects; FO modulated the expression of 7 genes, demonstrating an antiapoptotic effect. Conclusion: In patients with gastrointestinal cancer, preoperative MCT/LCT infusion was associated with postoperative lymphocyte and neutrophil apoptosis. FO has a protective effect on postoperative lymphocyte apoptosis. (JPEN J Parenter Enteral Nutr. 2012; 36: 677-684)