alpha-Tocopherol protects against alpha-naphthylisothiocyanate-induced hepatotoxicity in rats less effectively than melatonin

The protective effect of alpha-tocopherol (alpha-Toc), which exerts antioxidant and anti-inflammatory actions, against alpha-naphthylisothiocyanate (ANIT)-induced hepatotoxicity in rats was compared with that of melatonin because orally administered melatonin is known to protect against ANIT-induced hepatotoxicity in rats through its antioxidant and anti-inflammatory actions. Rats intoxicated once with ANIT (75 mg/kg, intraperitoneal (i.p.)) showed liver cell damage and biliary cell damage with cholestasis at 24 h, but not 12 h, after intoxication. ANIT-intoxicated rats received alpha-Toc (100 or 250 mg/kg) or melatonin (100 mg/kg) orally at 12 h after intoxication. The alpha-Toc administration protected against liver cell damage in ANIT-intoxicated rats, while the melatonin administration protected against both liver cell damage and biliary cell damage with cholestasis. ANIT-intoxicated rats had increased hepatic lipid peroxide concentration and myeloperoxidase activity at 12 and 24 h after intoxication. ANIT-intoxicated rats also had increased serum alpha-Toc and non-esterified fatty acid (NEFA) concentrations at 12 and 24 h after intoxication and increased serum triglyceride and total cholesterol concentrations at 24h. The administration of alpha-Toc to ANIT-intoxicated rats increased the hepatic alpha-Toc concentration with further increase in the serum alpha-Toc concentration and attenuated the increased hepatic lipid peroxide concentration and myeloperoxidase activity and serum NEFA concentration at 24 h after intoxication. The melatonin administration did not affect the hepatic alpha-Toc concentration but attenuated the increased hepatic lipid peroxide concentration and myeloperoxidase activity and serum alpha-Toc, NEFA, triglyceride, and total cholesterol concentrations at 24 h after ANIT intoxication. These results indicate that orally administered alpha-Toc protects against ANIT-induced hepatotoxicity in rats possibly through its antioxidant and anti-inflammatory actions less effectively than orally administered melatonin.     

Metabolic regulation of neutrophil spreading, membrane tubulovesicular extensions (cytonemes) formation and intracellular pH upon adhesion to fibronectin

Circulating leukocytes have a round cell shape and roll along vessel walls. However, metabolic disorders can lead them to adhere to the endothelium and spread (flatten). We studied the metabolic regulation of adhesion, spreading and intracellular pH (pHi) of neutrophils (polymorphonuclear leukocytes) upon adhesion to fibronectin-coated substrata. Resting neutrophils adhered and spread on fibronectin. An increase in pHi accompanied neutrophil spreading. Inhibition of oxidative phosphorylation or inhibition of P- and F-type ATPases affected neither neutrophil spreading nor pHi. Inhibition of glucose metabolism or V-ATPase impaired neutrophil spreading, blocked the increase in the pHi and induced extrusion of membrane tubulovesicular extensions (cytonemes), anchoring cells to substrata. Omission of extracellular Na(+) and inhibition of chloride channels caused a similar effect. We propose that these tubulovesicular extensions represent protrusions of exocytotic trafficking, supplying the plasma membrane of neutrophils with ion exchange mechanisms and additional membrane for spreading. Glucose metabolism and V-type ATPase could affect fusion of exocytotic trafficking with the plasma membrane, thus controlling neutrophil adhesive state and pHi. Cl(-) efflux through chloride channels and Na(+) influx seem to be involved in the regulation of the V-ATPase by carrying out charge compensation for the proton-pumping activity and through V-ATPase in regulation of neutrophil spreading and pHi.     

Neutrophil elastase sorting involves plasma membrane trafficking requiring the C-terminal propeptide

The primary granules/secretory lysosomes of neutrophils store mature neutrophil elastase (NE) as a luminal protein after proteolytic removal of N-terminal and C-terminal pro-peptides from a proform of NE. The N-terminal pro-peptide prevents premature activation that might be toxic to the cell, but the C-terminal pro-peptide has no defined function. In this study, we investigated the role of the C-terminal pro-peptide in trafficking of NE by expressing, in rat basophilic leukemia (RBL) cells, both wild-type NE and the mutant NE/Delta248-267, which lacks the C-terminal pro-peptide. Both transfected proteins were found to be targeted to secretory lysosomes. In addition, results from antibody ligation and cell-surface biotinylation indicated that proform of NE was targeted to the plasma membrane, and then subjected to endocytosis. The results were supported by the detection of targeting of the proform to the plasma membrane followed by internalization both in RBL cells and normal granulopoietic precursor cells. Targeting of NE to the plasma membrane required the C-terminal pro-peptide as NE/Delta248-267 expressed in RBL cells bypassed plasma membrane trafficking. Our results indicate targeting of a population of NE to the plasma membrane and internalization dependent on the C-terminal NE pro-peptide.     

Effects of microcystins on human polymorphonuclear leukocytes

Microcystins (MCs) are cyclic heptapeptides produced by cyanobacteria present in water contaminated reservoirs. Reported toxic effects for microcystins are liver injury and tumour promotion. In this study, we evaluated the effects of two MCs, MC-LR and [Asp(3)]-MC-LR, on human neutrophil (PMN). We observed that even at concentrations lower than that recommended by World Health Organization for chronic exposure (0.1 nM), MCs affect human PMN. Both MCs have chemotactic activity, induce the production of reactive oxygen species, and increase phagocytosis of Candida albicans. MC-LR also increased C. albicans killing. The effect of MCs on PMN provides support for a damage process mediated by PMN and oxidative stress, and may explain liver injury and tumour promotion associated to long-term MCs exposures.     

Src homology 2 domain of overexpressed Lyn kinase is responsible for the acceleration of granulocyte colony-stimulating factor-induced neutrophilic nuclear lobulation

We have previously shown that the overexpression of a Src family kinase, Lyn, and its kinase-negative form, LynKN, in a granulocyte progenitor cell line, GM-I62M, accelerates neutrophilic nuclear lobulation when the cells are cultured in the presence of granulocyte colony-stimulating factor. In this study, we investigated the role of the Src homology 2 (SH2) and SH3 domains of Lyn in the accelerated induction of nuclear lobulation. In contrast to wild-type Lyn, the overexpression of its SH2 domain mutant did not induce the accelerated nuclear morphological changes, but the overexpressed SH3 domain mutant had the same effects as wild-type Lyn. Therefore, the SH2 domain of Lyn is responsible for the accelerated induction of neutrophilic nuclear lobulation upon G-CSF stimulation.     

Signal-dependent slow leukocyte rolling does not require cytoskeletal anchorage of P-selectin glycoprotein ligand-1 (PSGL-1) or integrin αLβ2

In inflamed venules, neutrophils roll on P- or E-selectin, engage P-selectin glycoprotein ligand-1 (PSGL-1), and signal extension of integrin α(L)β(2) in a low affinity state to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Cytoskeleton-dependent receptor clustering often triggers signaling, and it has been hypothesized that the cytoplasmic domain links PSGL-1 to the cytoskeleton. Chemokines cause rolling neutrophils to fully activate α(L)β(2), leading to arrest on ICAM-1. Cytoskeletal anchorage of α(L)β(2) has been linked to chemokine-triggered extension and force-regulated conversion to the high affinity state. We asked whether PSGL-1 must interact with the cytoskeleton to initiate signaling and whether α(L)β(2) must interact with the cytoskeleton to extend. Fluorescence recovery after photobleaching of transfected cells documented cytoskeletal restraint of PSGL-1. The lateral mobility of PSGL-1 similarly increased by depolymerizing actin filaments with latrunculin B or by mutating the cytoplasmic tail to impair binding to the cytoskeleton. Converting dimeric PSGL-1 to a monomer by replacing its transmembrane domain did not alter its mobility. By transducing retroviruses expressing WT or mutant PSGL-1 into bone marrow-derived macrophages from PSGL-1-deficient mice, we show that PSGL-1 required neither dimerization nor cytoskeletal anchorage to signal β(2) integrin-dependent slow rolling on P-selectin and ICAM-1. Depolymerizing actin filaments or decreasing actomyosin tension in neutrophils did not impair PSGL-1- or chemokine-mediated integrin extension. Unlike chemokines, PSGL-1 did not signal cytoskeleton-dependent swing out of the β(2)-hybrid domain associated with the high affinity state. The cytoskeletal independence of PSGL-1-initiated, α(L)β(2)-mediated slow rolling differs markedly from the cytoskeletal dependence of chemokine-initiated, α(L)β(2)-mediated arrest.     

Apolipoproteins and Apolipoprotein Mimetic Peptides Modulate Phagocyte Trafficking through Chemotactic Activity

The plasma lipoprotein-associated apolipoproteins (apo) A-I and apoE have well described anti-inflammatory actions in the cardiovascular system, and mimetic peptides that retain these properties have been designed as therapeutics. The anti-inflammatory mechanisms of apolipoprotein mimetics, however, are incompletely defined. Whether circulating apolipoproteins and their mimetics regulate innate immune responses at mucosal surfaces, sites where transvascular emigration of leukocytes is required during inflammation, remains unclear. Herein, we report that Apoai^−/− and Apoe^−/− mice display enhanced recruitment of neutrophils to the airspace in response to both inhaled lipopolysaccharide and direct airway inoculation with CXCL1. Conversely, treatment with apoA-I (L-4F) or apoE (COG1410) mimetic peptides reduces airway neutrophilia. We identify suppression of CXCR2-directed chemotaxis as a mechanism underlying the apolipoprotein effect. Pursuing the possibility that L-4F might suppress chemotaxis through heterologous desensitization, we confirmed that L-4F itself induces chemotaxis of human PMNs and monocytes. L-4F, however, fails to induce a calcium flux. Further exploring structure-function relationships, we studied the alternate apoA-I mimetic L-37pA, a bihelical analog of L-4F with two Leu-Phe substitutions. We find that L-37pA induces calcium and chemotaxis through formyl peptide receptor (FPR)2/ALX, whereas its D-stereoisomer (i.e. D-37pA) blocks L-37pA signaling and induces chemotaxis but not calcium flux through an unidentified receptor. Taken together, apolipoprotein mimetic peptides are novel chemotactic agents that possess complex structure-activity relationships to multiple receptors, displaying anti-inflammatory efficacy against innate immune responses in the airway.     

Surfactant protein D (Sp-D) binds to membrane-proximal domain (D3) of signal regulatory protein α (SIRPα), a site distant from binding domain of CD47, while also binding to analogous region on signal regulatory protein β (SIRPβ)

Signal regulatory protein α (SIRPα), a highly glycosylated type-1 transmembrane protein, is composed of three immunoglobulin-like extracellular loops as well as a cytoplasmic tail containing three classical tyrosine-based inhibitory motifs. Previous reports indicate that SIRPα binds to humoral pattern recognition molecules in the collectin family, namely surfactant proteins D and A (Sp-D and Sp-A, respectively), which are heavily expressed in the lung and constitute one of the first lines of innate immune defense against pathogens. However, little is known about molecular details of the structural interaction of Sp-D with SIRPs. In the present work, we examined the molecular basis of Sp-D binding to SIRPα using domain-deleted mutant proteins. We report that Sp-D binds to the membrane-proximal Ig domain (D3) of SIRPα in a calcium- and carbohydrate-dependent manner. Mutation of predicted N-glycosylation sites on SIRPα indicates that Sp-D binding is dependent on interactions with specific N-glycosylated residues on the membrane-proximal D3 domain of SIRPα. Given the remarkable sequence similarity of SIRPα to SIRPβ and the lack of known ligands for the latter, we examined Sp-D binding to SIRPβ. Here, we report specific binding of Sp-D to the membrane-proximal D3 domain of SIRPβ. Further studies confirmed that Sp-D binds to SIRPα expressed on human neutrophils and differentiated neutrophil-like cells. Because the other known ligand of SIRPα, CD47, binds to the membrane-distal domain D1, these findings indicate that multiple, distinct, functional ligand binding sites are present on SIRPα that may afford differential regulation of receptor function.     

Neutrophil elastase, proteinase 3 and cathepsin G: physicochemical properties, activity and physiopathological functions

Polymorphonuclear neutrophils form a primary line of defense against bacterial infections using complementary oxidative and non-oxidative pathways to destroy phagocytized pathogens. The three serine proteases elastase, proteinase 3 and cathepsin G, are major components of the neutrophil primary granules that participate in the non-oxidative pathway of intracellular pathogen destruction. Neutrophil activation and degranulation results in the release of these proteases into the extracellular medium as proteolytically active enzymes, part of them remaining exposed at the cell surface. Extracellular neutrophil serine proteases also help kill bacteria and are involved in the degradation of extracellular matrix components during acute and chronic inflammation. But they are also important as specific regulators of the immune response, controlling cellular signaling through the processing of chemokines, modulating the cytokine network, and activating specific cell surface receptors. Neutrophil serine proteases are also involved in the pathogenicity of a variety of human diseases. This review focuses on the structural and functional properties of these proteases that may explain their specific biological roles, and facilitate their use as molecular targets for new therapeutic strategies.     

Preparation of Secretory Vesicle-Free Plasma Membranes by Isopycnic Sucrose Gradient Fractionation of Neutrophils Purified by the Gelatin Method

Isolated human neutrophils serve as a model for the in vitro study of host defensive processes as well as the cell biology and biochemistry of primary human cells. We demonstrate that the requirements of the gelatinbased procedure for neutrophil isolation from whole blood induces the complete loss of secretory vesicles from in vitro isolated populations, whereas isolation by a dextran-based methodology results in the preservation of this organelle. Following density fractionation of cellular cavitates, examination of commonly employed plasma membrane marker activities yielded subcellular localization patterns that were indistinguishable between dextran- or gelatin-isolated populations, indicating both populations to be otherwise comparable in terms of the relative complexity and large-scale organization of plasma membranes. Given that the cell surface upregulation of secretory vesicles is implicated as an initial requirement of neutrophil activation as well as an intrinsic feature of neutrophil priming, we show that dextran and gelatin-isolated neutrophils may be considered to occupy functionally nonactivated and primed cellular states, respectively. These differences in phenotype can be exploited in specific ways. We suggest that the gelatin method has technical advantages with regard to the study of neutrophil plasma membranes. In particular, results from this study indicate the gelatin method to be a reliable and effective preparatory technique appropriate for tandem use with density fractionation procedures to achieve rapid isolation of plasma membranes that are uncontaminated by secretory organelles.