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1.
J Biol Chem ; 292(37): 15312-15320, 2017 09 15.
Article in English | MEDLINE | ID: mdl-28747436

ABSTRACT

The immunomodulatory receptor Siglec-3/CD33 influences risk for late-onset Alzheimer's disease (LOAD), an apparently human-specific post-reproductive disease. CD33 generates two splice variants: a full-length CD33M transcript produced primarily by the "LOAD-risk" allele and a shorter CD33m isoform lacking the sialic acid-binding domain produced primarily from the "LOAD-protective" allele. An SNP that modulates CD33 splicing to favor CD33m is associated with enhanced microglial activity. Individuals expressing more protective isoform accumulate less brain ß-amyloid and have a lower LOAD risk. How the CD33m isoform increases ß-amyloid clearance remains unknown. We report that the protection by the CD33m isoform may not be conferred by what it does but, rather, from what it cannot do. Analysis of blood neutrophils and monocytes and a microglial cell line revealed that unlike CD33M, the CD33m isoform does not localize to cell surfaces; instead, it accumulates in peroxisomes. Cell stimulation and activation did not mobilize CD33m to the surface. Thus, the CD33m isoform may neither interact directly with amyloid plaques nor engage in cell-surface signaling. Rather, production and localization of CD33m in peroxisomes is a way of diminishing the amount of CD33M and enhancing ß-amyloid clearance. We confirmed intracellular localization by generating a CD33m-specific monoclonal antibody. Of note, CD33 is the only Siglec with a peroxisome-targeting sequence, and this motif emerged by convergent evolution in toothed whales, the only other mammals with a prolonged post-reproductive lifespan. The CD33 allele that protects post-reproductive individuals from LOAD may have evolved by adaptive loss-of-function, an example of the less-is-more hypothesis.


Subject(s)
Alzheimer Disease/genetics , Genetic Predisposition to Disease , Macrophages/metabolism , Microglia/metabolism , Neutrophils/metabolism , Polymorphism, Single Nucleotide , Sialic Acid Binding Ig-like Lectin 3/metabolism , Alleles , Alzheimer Disease/immunology , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amino Acid Motifs , Bacterial Proteins/metabolism , Bacterial Proteins/toxicity , Cell Line , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Membrane/pathology , Humans , Lipopolysaccharides/toxicity , Macrophage Activation/drug effects , Macrophages/drug effects , Macrophages/immunology , Macrophages/pathology , Microglia/cytology , Microglia/immunology , Microglia/pathology , N-Formylmethionine Leucyl-Phenylalanine/toxicity , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neuraminidase/metabolism , Neuraminidase/toxicity , Neutrophil Activation/drug effects , Neutrophils/drug effects , Neutrophils/immunology , Neutrophils/pathology , Peroxisomes/drug effects , Peroxisomes/metabolism , Peroxisomes/pathology , Phylogeny , Protein Interaction Domains and Motifs , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Sorting Signals , Protein Transport/drug effects , Sialic Acid Binding Ig-like Lectin 3/chemistry , Sialic Acid Binding Ig-like Lectin 3/genetics
2.
Pharm Biol ; 53(11): 1661-70, 2015.
Article in English | MEDLINE | ID: mdl-25856712

ABSTRACT

CONTEXT: Polymorphonuclear leukocytes (PMNs) produce oxidants, contributing to systemic oxidative stress. Diets rich in plant polyphenols seem to decrease the risk of oxidative stress-induced disorders including cardiovascular disease. OBJECTIVE: The objective of this study was to examine the in vitro effect of each of the 14 polyphenols on PMNs chemotaxis, intracellular calcium response, oxidants production. MATERIALS AND METHODS: Blood samples and PMNs suspensions were obtained from 60 healthy non-smoking donors and incubated with a selected polyphenol (0.5-10 µM) or a control solvent. We assessed resting and fMLP-dependent changes of intracellular calcium concentration ([Ca(2+)]i) in PMNs with the Fura-2AM method and measured fMLP-induced luminol enhanced whole blood chemiluminescence (fMLP-LBCL). Polyphenol chemoattractant activity for PMNs was tested with Boyden chambers. RESULTS: Polyphenols had no effect on resting [Ca(2+)]i. Unaffected by other compounds, fMLP-dependent increase of [Ca(2+)]i was inhibited by quercetin and catechol (5 µM) by 32 ± 14 and 12 ± 10% (p < 0.04), respectively. Seven of the 14 tested substances (5 µM) influenced fMLP-LBCL by decreasing it. Catechol, quercetin, and gallic acid acted most potently reducing fMLP-LBCL by 49 ± 5, 42 ± 15, and 28 ± 18% (p < 0.05), respectively. 3,4-Dihydroxyhydrocinnamic, 3,4-dihydroxyphenylacetic, 4-hydroxybenzoic acid, and catechin (5 µM) revealed distinct (p < 0.02) chemoattractant activity with a chemotactic index of 1.9 ± 0.8, 1.8 ± 0.7, 1.6 ± 0.6, 1.4 ± 0.2, respectively. CONCLUSION AND DISCUSSION: Catechol, quercetin, and gallic acid at concentrations commensurate in human plasma strongly suppressed the oxidative response of PMNs. Regarding quercetin and catechol, this could result from an inhibition of [Ca(2+)]i response.


Subject(s)
Calcium/metabolism , Chemotaxis/physiology , Luminescence , N-Formylmethionine Leucyl-Phenylalanine/toxicity , Neutrophils/metabolism , Phenols/pharmacology , Adult , Chemotaxis/drug effects , Female , Humans , Intracellular Fluid/drug effects , Intracellular Fluid/metabolism , Male , Neutrophils/drug effects , Phenols/isolation & purification , Plant Extracts/isolation & purification , Plant Extracts/pharmacology
3.
Toxicol Lett ; 221(1): 57-63, 2013 Jul 31.
Article in English | MEDLINE | ID: mdl-23726862

ABSTRACT

Inflammation is frequently associated with nanoparticle (NP) exposures. Given that excessive polymorphonuclear neutrophil cell degranulation is a common feature of inflammatory disorders, and since these cells are key players in inflammation, we decided to test the hypothesis that NPs could act as modulators of degranulation in human neutrophils. TiO2, CeO2 and ZnO NPs slightly down-regulated cell surface expression of the granule marker CD35, but increased CD66b and CD63 expression, as assessed by flow cytometry. In addition, expression of myeloperoxidase, MMP-9 and albumin stored in azurophil, specific/gelatinase and secretrory granules, respectively, was significantly increased in the supernatants of NPs-induced neutrophils when compared to untreated cells. Moreover, NPs were more potent than the classical bacterial tripeptide N-formyl-methionine-leucine-phenylalanine (fMLP) agonist. Finally, TiO2 and CeO2 markedly increased the enzymatic activity of MMP-9 released into the supernatant, as assessed by gelatin zymography, while ZnO exerted only a modest effect. We conclude that NPs can differentially affect all steps involved during neutrophil degranulation, namely, cell surface expression of granule markers, liberation of proteins in the supernatants and enzymatic activity. These results are expected to be helpful to understand the toxicity of TiO2, CeO2 and ZnO.


Subject(s)
Cell Degranulation/drug effects , Cerium/toxicity , Metal Nanoparticles/toxicity , Neutrophils/drug effects , Titanium/toxicity , Zinc Oxide/toxicity , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/metabolism , Albumins/metabolism , Cell Survival/drug effects , Cells, Cultured , Cytoplasmic Granules/drug effects , Cytoplasmic Granules/metabolism , Humans , Matrix Metalloproteinase 9/metabolism , N-Formylmethionine Leucyl-Phenylalanine/toxicity , Neutrophils/physiology , Particle Size , Peroxidase/metabolism , Polymerization
4.
J Immunol ; 183(11): 7557-68, 2009 Dec 01.
Article in English | MEDLINE | ID: mdl-19917697

ABSTRACT

Hepatic neutrophil adhesion during endotoxemia is an integrin-independent, CD44-dependent process. Because integrins function in other endotoxemic vasculatures, we used spinning disk confocal intravital microscopy to assess whether LPS down-modulated integrin functions in sinusoids. First, we applied fMLP onto the liver surface, and compared it with systemic LPS administration. Local fMLP caused neutrophil adhesion, crawling, and emigration for at least 2 h. Surprisingly, the number of adherent and crawling neutrophils was markedly reduced in Mac-1(-/-) and ICAM-1(-/-) mice, but not in mice treated with anti-CD44 mAb. By contrast, systemic LPS injection induced a robust accumulation of neutrophils in sinusoids, which was dependent on CD44, but not on integrins. Strikingly, local fMLP could not induce any integrin-dependent adhesion in endotoxemic mice treated with anti-CD44 mAb, indicating that Mac-1-dependent neutrophil adhesion was inhibited by LPS. This response was localized to the hepatic microvasculature because neutrophils still adhered via integrins in brain microvasculature. ICAM-1/ICAM-2 levels were not decreased, but following LPS treatment, Mac-1 was down-regulated in neutrophils localized to liver, but not in the circulation. Mac-1 down-regulation in neutrophils was not observed in IL-10(-/-) mice. In vitro neutrophil incubation with IL-10 induced direct decrease of Mac-1 expression and adhesivity in LPS-stimulated neutrophils. Therefore, our data suggest that Mac-1 is necessary for neutrophil adhesion and crawling during local inflammatory stimuli in sinusoids, but during systemic inflammation, neutrophils are exposed to high concentrations of IL-10, leading to a CD44-dependent, integrin-independent adhesion. This may be a mechanism to keep neutrophils in sinusoids for intravascular trapping.


Subject(s)
Endotoxemia/immunology , Interleukin-10/immunology , Liver/blood supply , Macrophage-1 Antigen/immunology , Microcirculation/immunology , Neutrophil Infiltration/immunology , Animals , Brain/blood supply , Brain/immunology , Cell Adhesion/immunology , Cell Adhesion Molecules/immunology , Cell Adhesion Molecules/metabolism , Down-Regulation , Endotoxemia/metabolism , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Hyaluronan Receptors/immunology , Hyaluronan Receptors/metabolism , Inflammation/immunology , Inflammation/metabolism , Interleukin-10/metabolism , Lipopolysaccharides/immunology , Lipopolysaccharides/toxicity , Liver/immunology , Macrophage-1 Antigen/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Confocal , N-Formylmethionine Leucyl-Phenylalanine/analogs & derivatives , N-Formylmethionine Leucyl-Phenylalanine/immunology , N-Formylmethionine Leucyl-Phenylalanine/toxicity , Neutrophils/immunology , Neutrophils/metabolism
5.
J Immunol ; 183(2): 1413-8, 2009 Jul 15.
Article in English | MEDLINE | ID: mdl-19564343

ABSTRACT

VAMP8, a member of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) family of fusion proteins, initially characterized in endosomal and endosomal-lysosomal fusion, may also function in regulated exocytosis. VAMP8 physiological function in inflammation has not been elucidated. In this paper, we show that deficiency of VAMP8 protects mice from anaphylatoxin (C5a)-induced neutropenia, peritonitis, and systemic inflammation. We show that, in vivo, VAMP8 deletion inhibits neutropenia and phagocyte recruitment. We also show that in macrophages, VAMP8 localizes on secretory granules and degranulation is inhibited in VAMP8-deficient macrophages. Moreover, VAMP8(-/-) mice show reduced systemic inflammation with inhibition of serum TNF-alpha levels, whereas IL-1beta, IL-6, and MIP1alpha release are not affected. In wild-type macrophages, TNF-alpha colocalizes with VAMP8-positive vesicles, and in VAMP8-deficient macrophages, the TNF-alpha release is inhibited. Furthermore, VAMP8 regulates the release of TNF-alpha and beta-hexosaminidase triggered by fMLP, and VAMP8(-/-) mice are protected from fMLP-induced peritonitis. These data demonstrate that the VAMP8 vesicle-associated-SNARE is required for the proper trafficking of secretory lysosomal granules for exocytosis in macrophages and for the release of the potent proinflammatory cytokine, TNF-alpha.


Subject(s)
Anaphylatoxins/pharmacology , Cell Degranulation/drug effects , R-SNARE Proteins/physiology , Tumor Necrosis Factor-alpha/metabolism , Animals , Cytokines/blood , Exocytosis , Immunologic Factors , Inflammation , Macrophages , Mice , Mice, Knockout , N-Formylmethionine Leucyl-Phenylalanine/toxicity , Neutropenia , Peritonitis/chemically induced , Phagocytes , R-SNARE Proteins/deficiency , Secretory Vesicles
6.
Am J Respir Cell Mol Biol ; 41(6): 680-7, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19307611

ABSTRACT

The morphological features of chronic obstructive pulmonary disease in man include emphysema and chronic bronchitis associated with mucus hypersecretion. These alterations can be induced in mice by a single intratracheal instillation of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), a chemoattractant and degranulating agent for neutrophils. The mechanisms underlying excessive mucus production and, in particular, goblet cell hyperplasia/metaplasia in chronic obstructive pulmonary disease remain poorly understood. The proteinase-activated receptors (PARs) are widely recognized for their modulatory properties during inflammation. In this study, we examined whether PAR-1 contributes to inflammation and lung damage induced by fMLP by comparing the response of PAR-1-deficient (PAR-1(-/-)) mice with that of wild-type (WT) mice. Mice were killed at various time points after fMLP instillation (200 microg/50 microl). WT mice developed emphysema and goblet cell metaplasia. The onset of pulmonary lesions was preceded by an increase in thrombin immunoreactivity in bronchial airways and alveolar tissue. This was followed by a decrease in PAR-1 immunoreactivity, and by an increase in IL-13 immunostaining on the luminal surface of airway epithelial cells. In PAR-1(-/-) mice, fMLP administration induced similar responses in terms of inflammation and emphysema, but these mice were protected from the development of goblet cell metaplasia. The involvement of PAR-1 in airway epithelial cell transdifferentiation was confirmed by demonstrating that intratracheal instillation of the selective PAR-1 agonist (TFLLR) induced goblet cell metaplasia in the airways of WT mice only. These data suggest that emphysema and goblet cell metaplasia occur independently, and that PAR-1 signaling through IL-13 stimulation may play an important role in inducing goblet cell metaplasia.


Subject(s)
Goblet Cells/drug effects , Goblet Cells/metabolism , N-Formylmethionine Leucyl-Phenylalanine/toxicity , Receptor, PAR-1/deficiency , Animals , Cell Differentiation/drug effects , Emphysema/chemically induced , Emphysema/metabolism , Emphysema/pathology , ErbB Receptors/metabolism , Goblet Cells/pathology , Humans , Interleukin-13/metabolism , Lung/drug effects , Lung/metabolism , Lung/pathology , Male , Metaplasia , Mice , Mice, Inbred C57BL , Mice, Knockout , Oligopeptides/pharmacology , Pulmonary Disease, Chronic Obstructive/etiology , Receptor, PAR-1/agonists , Receptor, PAR-1/genetics , Signal Transduction
7.
Environ Health Perspect ; 116(5): 593-8, 2008 May.
Article in English | MEDLINE | ID: mdl-18470306

ABSTRACT

BACKGROUND: Parkinson disease (PD), a chronic neurodegenerative disease, has been proposed to be a multifactorial disorder resulting from a combination of environmental mechanisms (chemical, infectious, and traumatic), aging, and genetic deficits. Microglial activation is important in the pathogenesis of PD. OBJECTIVES: We investigated dopaminergic (DA) neurotoxicity and the underlying mechanisms of formyl-methionyl-leucyl-phenylalanine (fMLP), a bacteria-derived peptide, in relation to PD. METHODS: We measured DA neurotoxicity using a DA uptake assay and immunocytochemical staining (ICC) in primary mesencephalic cultures from rodents. Microglial activation was observed via ICC, flow cytometry, and superoxide measurement. RESULTS: fMLP can cause selective DA neuronal loss at concentrations as low as 10(-13) M. Further, fMLP (10(-13) M) led to a significant reduction in DA uptake capacity in neuron/glia (N/G) cultures, but not in microglia-depleted cultures, indicating an indispensable role of microglia in fMLP-induced neurotoxicity. Using ICC of a specific microglial marker, OX42, we observed morphologic changes in activated microglia after fMLP treatment. Microglial activation after fMLP treatment was confirmed by flow cytometry analysis of major histocompatibility antigen class II expression on a microglia HAPI cell line. Mechanistic studies revealed that fMLP (10(-13) M)-induced increase in the production of extracellular superoxide from microglia is critical in mediating fMLP-elicited neurotoxicity. Pharmacologic inhibition of NADPH oxidase (PHOX) with diphenylene-iodonium or apocynin abolished the DA neurotoxicity of fMLP. N/G cultures from PHOX-deficient (gp91PHOX-/ -) mice were also insensitive to fMLP-induced DA neurotoxicity. CONCLUSION: fMLP (10(-13) M) induces DA neurotoxicity through activation of microglial PHOX and subsequent production of superoxide, suggesting a role of fMLP in the central nervous system inflammatory process.


Subject(s)
Central Nervous System Infections/etiology , Dopamine/metabolism , Microglia/drug effects , N-Formylmethionine Leucyl-Phenylalanine/toxicity , Neurodegenerative Diseases/chemically induced , Parkinson Disease/etiology , Animals , Cells, Cultured , Central Nervous System Infections/metabolism , Central Nervous System Infections/pathology , Enzyme Activation , Female , Mice , Mice, Inbred C57BL , Mice, Knockout , Microglia/enzymology , Microglia/metabolism , NADPH Oxidases/metabolism , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/pathology , Neurons/drug effects , Neurons/metabolism , Oxidative Stress , Pregnancy , Rats , Rats, Inbred F344
8.
Am J Respir Cell Mol Biol ; 39(1): 7-18, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18239191

ABSTRACT

The cyclin-dependent kinase inhibitor p21(CIP1/WAF1/SDI1) (p21) is an important inhibitory checkpoint regulator of cell cycle progression in response to oxidative and genotoxic stresses. It is known that p21 potentiates inflammatory response and inhibits apoptosis and proliferation, leading to cellular senescence. However, the role of endogenous p21 in regulation of lung inflammatory and injurious responses by cigarette smoke (CS) or other pro-inflammatory stimuli is not known. We hypothesized that p21 is an important modifier of lung inflammation and injury, and genetic ablation of p21 will confer protection against CS and other pro-inflammatory stimuli (lipopolysacchride [LPS] and N-formyl-methionyl-leucyl-phenylalanine [fMLP])-mediated lung inflammation and injury. To test this hypothesis, p21-deficient (p21-/-) and wild-type mice were exposed to CS, LPS, or fMLP, and the lung oxidative stress and inflammatory responses as well as airspace enlargement were assessed. We found that targeted disruption of p21 attenuated CS-, LPS-, or fMLP-mediated lung inflammatory responses in mice. CS-mediated oxidative stress and fMLP-induced airspace enlargement were also decreased in lungs of p21-/- mice compared with wild-type mice. The mechanism underlying this finding was associated with decreased NF-kappaB activation, and reactive oxygen species generation by decreased phosphorylation of p47(phox) and down-modulating the activation of p21-activated kinase. Our data provide insight into the mechanism of pro-inflammatory effect of p21, and the loss of p21 protects against lung oxidative and inflammatory responses, and airspace enlargement in response to multiple pro-inflammatory stimuli. These data may have ramifications in CS-induced senescence in the pathogenesis of chronic obstructive pulmonary disease/emphysema.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/deficiency , Cyclin-Dependent Kinase Inhibitor p21/physiology , Inflammation/etiology , Inflammation/prevention & control , Lipopolysaccharides/toxicity , Lung/physiopathology , N-Formylmethionine Leucyl-Phenylalanine/toxicity , Smoke/adverse effects , Aerosols , Animals , Bronchoalveolar Lavage , Cell Cycle/physiology , Crosses, Genetic , Cyclin-Dependent Kinase Inhibitor p21/genetics , Inflammation/chemically induced , Inflammation/pathology , Lipopolysaccharides/adverse effects , Mice , Mice, Inbred C57BL , Mice, Knockout , Peroxidase/metabolism
9.
BMC Immunol ; 8: 31, 2007 Nov 28.
Article in English | MEDLINE | ID: mdl-18045459

ABSTRACT

BACKGROUND: During inflammation, beta2-integrins mediate leukocyte adhesion to the endothelium accompanied by the activation of the spleen tyrosine kinase Syk. RESULTS: We investigated leukocyte adhesion and rolling in cremaster muscle venules before and during stimulation with fMLP using mice with a Syk-/- hematopoietic system. In unstimulated venules, Syk-/- leukocytes adhered less efficiently than control leukocytes while rolling was similar between Syk-/- and control leukocytes. During fMLP-superfusion, control mice showed significantly increased adhesion accompanied by reduced rolling. For Syk-/- leukocytes, an increase in adhesion with a concomitant decrease in rolling was only observed during the first three minutes during fMLP stimulation, but not at later time points. We also investigated leukocyte spreading against the vessel wall during fMLP stimulation and found a significant impairment of spreading for Syk-/- leukocytes. Additional in vitro experiments revealed that the adhesion and spreading defect seen in Syk-/- chimeric mice was due to compromised beta2-integrin-mediated outside-in signaling. CONCLUSION: We provide substantial evidence for an important role of Syk in mediating beta2-integrin dependent outside-in signaling leading to sustained leukocyte adhesion and spreading during the inflammatory response in vivo.


Subject(s)
Cell Adhesion/immunology , Inflammation/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Leukocyte Rolling/physiology , Protein-Tyrosine Kinases/metabolism , Animals , CD18 Antigens/immunology , CD18 Antigens/metabolism , Endothelium, Vascular/immunology , Endothelium, Vascular/metabolism , Image Processing, Computer-Assisted , Inflammation/chemically induced , Inflammation/immunology , Intracellular Signaling Peptides and Proteins/immunology , Mice , Mice, Mutant Strains , Muscle, Skeletal/blood supply , N-Formylmethionine Leucyl-Phenylalanine/toxicity , Protein-Tyrosine Kinases/immunology , Syk Kinase , Transplantation Chimera , Venules/immunology
11.
J Thromb Haemost ; 1(2): 314-9, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12871506

ABSTRACT

Epidemiological studies link acute infection of the respiratory tract to a transient increased risk of acute myocardial infarction. The underlying mechanisms remain unknown. We hypothesized that vasoactive mediators produced by inflammatory cells in the lungs and drained in the coronary circulation may trigger acute myocardial ischemia. To test this hypothesis we used an experimental model in the rabbit. Injection of the bacterial-derived peptide N-formyl-Met-Leu-Phe (or N-formyl-Methionyl-Leucyl-Phenylalanine)(fMLP) in the jugular vein induced massive recruitment of both polymorphonuclear leukocytes (PMN) and platelets in the microcirculation of the lungs, accompanied by rapid and marked increase of leukotriene B4, cysteinyl leukotrienes and thromboxane (Tx) A2 in the aortic blood. In all animals, fMLP evoked ischemic electrocardiographic changes: within the first minute of infusion a profound depression of the ST segment and inversion of the T wave were observed. Mean aortic pressure and heart rate fell to 64.0 +/- 6.9 and 83.5 +/- 3.1% of the basal levels at 3 and 10 min, respectively. All these alterations were transient. Aspirin, prevented electrocardiographic ischemic changes, reverted bradycardia and hypotension but did not significantly modify either PMN or platelet recruitment nor leukotriene synthesis. Ridogrel, a Tx-synthase and receptor inhibitor, prevented ECG alterations and bradycardia, but did not prevent and even worsened hypotension; it blocked platelet, but not PMN, sequestration. Pretreatment of animals with intravenous high dose of aspirin prevented ridogrel-dependent hypotension and platelet inhibition, suggesting that PGI2 contributes to the effects of Tx-synthase and receptor inhibitor. In hypercholesterolemic rabbits, ECG alterations persisted longer than in normal controls. In summary, our results indicate that acute activation of PMN and platelets in the lungs provokes transient myocardial ischemia, in normal animals that is exacerbated in hypercholesterolemic rabbits. TxA2 appears to be the major mediator of this phenomenon. Moreover the data suggest that a balance between TxA2 and PGI2 plays a pivotal role in platelet activation and recruitment in our model.


Subject(s)
Myocardial Ischemia/etiology , Myocardial Ischemia/physiopathology , Pneumonia/complications , Pneumonia/physiopathology , Thromboxane A2/physiology , Acute Disease , Animals , Arteriosclerosis/blood , Arteriosclerosis/complications , Arteriosclerosis/physiopathology , Disease Models, Animal , Electrocardiography , Epoprostenol/physiology , Inflammation Mediators/physiology , Male , Myocardial Ischemia/blood , N-Formylmethionine Leucyl-Phenylalanine/toxicity , Platelet Activation/physiology , Pneumonia/blood , Rabbits
12.
Liver ; 19(3): 253-8, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10395046

ABSTRACT

BACKGROUND/AIMS: Cholangitis in rats induced by N-formyl L-methionine L-leucine L-tyrosine (fMLT) is characterized by infiltration of mononuclear cells around bile ducts in portal tracts. METHODS: We investigated the initial process in fMLT-induced cholangitis histochemically. RESULTS: Administration of fMLT into the colons of adult male Wistar rats with acetate-induced colitis resulted in an infiltration of mostly macrophages and granulocytes into the portal tracts on day 1. Abnormal peroxidation as demonstrated by the nitro blue tetrazolium (NBT) reaction occurred in bile duct cells as well, although no apparent necrosis of the bile duct cells was observed. On day 4, the majority of the inflammatory cells in the portal tracts were CD4+ or CD8+ T lymphocytes. The oxidative products of the NBT reaction also disappeared from the bile duct cells. Administration of carrageenan, a potent inhibitor of macrophage function, resulted in a significant decrease in lymphocyte infiltration into the portal tracts. On day 8, portal inflammation subsided. CONCLUSIONS: In formyl peptide-induced cholangitis, macrophages and granulocytes may injure bile ducts transiently. Further, macrophages are necessary for the subsequent migration of T lymphocytes around the bile ducts.


Subject(s)
Cell Movement/immunology , Cholangitis/immunology , Lymphocytes/immunology , Macrophages/immunology , N-Formylmethionine Leucyl-Phenylalanine/analogs & derivatives , Animals , CD4-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/cytology , Carrageenan/pharmacology , Cell Count , Cholangitis/chemically induced , Cholangitis/complications , Colitis/chemically induced , Colitis/complications , Immunohistochemistry , Indicators and Reagents , Liver/drug effects , Liver/pathology , Lymphocytes/cytology , Macrophages/cytology , Macrophages/drug effects , Male , N-Formylmethionine Leucyl-Phenylalanine/toxicity , Nitroblue Tetrazolium , Rats , Rats, Wistar , Time Factors
13.
J Pharmacol Exp Ther ; 282(2): 1005-10, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9262369

ABSTRACT

We evaluated the potency of truncated secretory leukoprotease inhibitor (truncated SLPI) in a human sputum elastase (HSE)-induced lung injury model and in a specific neutrophil-mediated acute lung injury model in hamsters. Intratracheal administration of HSE induced acute lung hemorrhage that could be measured by determination of the hemoglobin content in the bronchoalveolar lavage fluid. Intratracheal administration of truncated SLPI 1 hr before HSE administration inhibited acute lung hemorrhage in a dose-dependent manner (ED50 = 46.8 microg/kg), as did i.v. injection of the inhibitor given 2 min before HSE administration (ED50 = 14.7 mg/kg). Intratracheal administration of endotoxin (lipopolysaccharide) induced pulmonary neutrophilia. Twenty-four hours after lipopolysaccharide administration, the addition of formyl-methionyl-leucyl-phenylalanine resulted in a neutrophil-dependent acute lung injury that expressed an increase in hemoglobin content and in elastase-like activity in bronchoalveolar lavage fluids. In this model, lung injury was significantly attenuated by i.v. and intratracheal administration of truncated SLPI. These results suggest that truncated SLPI appears to be a good candidate inhibitor for the treatment of destructive lung diseases due to neutrophils.


Subject(s)
Lung Diseases/enzymology , Proteins/pharmacology , Animals , Bronchoalveolar Lavage Fluid , Cricetinae , Disease Models, Animal , Lipopolysaccharides/toxicity , Lung Diseases/chemically induced , Lung Diseases/pathology , Male , Mesocricetus , N-Formylmethionine Leucyl-Phenylalanine/toxicity , Pancreatic Elastase/toxicity , Proteinase Inhibitory Proteins, Secretory , Secretory Leukocyte Peptidase Inhibitor
14.
J Gastroenterol Hepatol ; 12(5): 360-7, 1997 May.
Article in English | MEDLINE | ID: mdl-9195381

ABSTRACT

Pathogenesis of gastric damage induced by non-steroidal anti-inflammatory drugs (NSAID) involves multiple elements, such as deficiency of prostaglandins (PG), gastric hypermotility, neutrophil activation and luminal acid. The present study was performed to examine the effects of these elements, either alone or in combination, on the rat gastric mucosa and investigate which element is most closely associated with the gastric ulcerogenic response to NSAID. The following treatments were used to express various pathogenic elements: (i) a low dose of indomethacin (IM) to cause PG deficiency; (ii) 2-deoxy-D-glucose (2DG) to induce gastric hypermotility and acid secretion; (iii) histamine to induce acid hypersecretion; and (iv) n-formyl-Met-Leu-Phe (fMLP) to elicit neutrophil activation. When rats fasted for 18 h were subjected to each treatment alone, only 2DG caused slight macroscopic damage in the gastric mucosa within 4 h. Indomethacin showed over 90% inhibition of mucosal PG generation and fMLP increased myeloperoxidase activity four-fold greater than normal values, yet either of these treatments alone did not cause any damage in the stomach. However, the combination of IM with 2DG or His provoked severe lesions in the stomach or the duodenum, respectively, while fMLP did not modify or potentiate the mucosal ulcerogenic response to other treatments. We conclude that among various pathogenic elements only gastric hypermotility is sufficient, by itself, to induce mild damage in the mucosa, that PG deficiency may be critical in the increase of mucosal susceptibility to injury and that neutrophil activation alone is not ulcerogenic in the gastric mucosa nor does it potentiate the ulcerogenic effect of other elements. Luminal acid may be a prerequisite for later extension of damage to severe lesions.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/toxicity , Gastric Mucosa/pathology , Indomethacin/toxicity , Stomach Ulcer/pathology , Animals , Antimetabolites/toxicity , Deoxyglucose/toxicity , Disease Models, Animal , Dose-Response Relationship, Drug , Gastric Acid/metabolism , Gastric Emptying/drug effects , Gastric Mucosa/drug effects , Gastric Mucosa/metabolism , Male , N-Formylmethionine Leucyl-Phenylalanine/toxicity , Neutrophil Activation/drug effects , Peroxidase/metabolism , Prostaglandins/deficiency , Prostaglandins/metabolism , Radioimmunoassay , Rats , Rats, Sprague-Dawley , Stomach Ulcer/chemically induced , Stomach Ulcer/metabolism
15.
Jpn J Pharmacol ; 70(4): 291-302, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8847836

ABSTRACT

The effects of the new anti-platelet agent KBT-3022, ethyl 2-[4,5-bis(4-methoxyphenyl)-thiazol-2-yl]pyrrol-1-ylacetate, and its metabolite desethyl KBT-3022 on rabbit neutrophil function were investigated in comparison with the effects of acetylsalicylic acid (ASA), ticlopidine hydrochloride (TP), cilostazol (CIL) and indomethacin (IM). The adhesion and migration of neutrophils induced by formyl-methionyl-leucyl-phenylalanine (fMLP) were inhibited by all the compounds tested, their rank order of potency being KBT-3022 = desethyl KBT-3022 > TP = CIL = IM > ASA. KBT-3022, desethyl KBT-3022, CIL and IM all suppressed fMLP-induced increases in the intracellular free Ca2+ concentration ([Ca2+]i) in neutrophils, their potencies correlating with their inhibitory effects on fMLP-induced adhesion and migration. KBT-3022 (1 microM), desethyl KBT-3022 (1-10 microM) and CIL (10 microM) but not IM significantly inhibited both neutrophil migration and the increase in [Ca2+]i induced by leukotriene B4 (LTB4). KBT-3022 (1 microM) and desethyl KBT-3022 (1 microM) suppressed the increase in [Ca2+]i induced by complement C5a. Although KBT-3022 and desethyl KBT-3022 did not influence [3H]LTB4 and [125I]C5a specific binding, [3H]fMLP specific binding was inhibited by desethyl KBT-3022 (IC50: 1.9 microM). Neutrophil adhesion and superoxide anion production stimulated by phorbol 12-myristate 13-acetate were partially inhibited by KBT-3022 (1 microM) and desethyl KBT-3022 (1-10 microM). These results suggest that KBT-3022 and desethyl KBT-3022 have a wider spectrum of action and are more potent inhibitors of neutrophil activation than ASA, TP, CIL and IM.


Subject(s)
Cyclooxygenase Inhibitors/pharmacology , Neutrophils/drug effects , Platelet Aggregation Inhibitors/pharmacology , Pyrroles/pharmacology , Thiazoles/pharmacology , Animals , Anti-Inflammatory Agents, Non-Steroidal/metabolism , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Aspirin/metabolism , Aspirin/pharmacology , Binding Sites , Calcium/metabolism , Cell Adhesion/drug effects , Cilostazol , Complement C3-C5 Convertases/metabolism , Complement C5a/metabolism , Complement C5a/toxicity , Cyclooxygenase Inhibitors/metabolism , Indomethacin/metabolism , Indomethacin/pharmacology , Leukotriene B4/metabolism , Leukotriene B4/toxicity , Male , N-Formylmethionine Leucyl-Phenylalanine/metabolism , N-Formylmethionine Leucyl-Phenylalanine/toxicity , Neutrophils/cytology , Neutrophils/metabolism , Platelet Activating Factor/metabolism , Platelet Activating Factor/toxicity , Platelet Aggregation Inhibitors/metabolism , Pyrroles/metabolism , Rabbits , Structure-Activity Relationship , Superoxides/metabolism , Tetradecanoylphorbol Acetate/toxicity , Tetrazoles/metabolism , Tetrazoles/pharmacology , Thiazoles/metabolism , Ticlopidine/metabolism , Ticlopidine/pharmacology
16.
Toxicology ; 104(1-3): 113-21, 1995 Dec 15.
Article in English | MEDLINE | ID: mdl-8560489

ABSTRACT

Aniline-denaturated rape-seed food oils that contained anilides of linoleic and oleic acids caused a poisoning epidemic, known as Toxic Oil Syndrome, in Spain in 1981. Toxic Oil Syndrome affected mainly the lungs and the immune system of exposed individuals. Linoleic and oleic acids, and linoleic and oleic anilides increased the production of reactive oxygen metabolites in human polymorphonuclear leukocytes. Both cis-fatty acids inhibited a chemotactic peptide-, fMLP-induced production of reactive oxygen metabolites without affecting fMLP-induced elevation of intracellular calcium levels. Linoleic acid anilide slightly amplified fMLP-induced respiratory burst, whereas oleic acid anilide was without an effect. However, both fatty acid anilides decreased fMLP-induced elevation of levels of free intracellular calcium. Moreover, both cis-fatty acids and their anilides inhibited phorbol myristate acetate (PMA)- and dioctanoyl-s,n-glycerol (DiC8)-induced production of reactive oxygen metabolites. Thus, both cis-fatty acids and their anilides inhibited agonist-stimulated production of reactive oxygen metabolites; this is most likely due to interactions with cell signalling events. These results suggest that both linoleic and oleic acids and their anilides may inhibit immunological responses of leukocytes.


Subject(s)
Anilides/toxicity , Linoleic Acids/toxicity , Neutrophils/drug effects , Oleic Acids/toxicity , Plant Oils/poisoning , Reactive Oxygen Species/metabolism , Analysis of Variance , Calcium/metabolism , Diglycerides/toxicity , Fatty Acids, Monounsaturated , Humans , Linoleic Acid , Linoleic Acids/metabolism , N-Formylmethionine Leucyl-Phenylalanine/toxicity , Neutrophils/metabolism , Oleic Acid , Oleic Acids/metabolism , Rapeseed Oil , Respiratory Burst/drug effects , Stereoisomerism , Tetradecanoylphorbol Acetate/toxicity
17.
Toxicology ; 100(1-3): 195-202, 1995 Jun 26.
Article in English | MEDLINE | ID: mdl-7624877

ABSTRACT

Human leukocytes were exposed to N-(5-vinyl-1,3-thiazolidin-2-ylidene)phenylamine (5-VTPA), a postulated impurity in the case oils that caused the Spanish Toxic Oil Syndrome in 1981. Changes induced by 5-VTPA alone and together with a chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP), a tumor promoter, phorbol myristate acetate (PMA), or a synthetic diacylglycerol, dioctanoyl-s,n-glycerol (DiC8) in free intracellular calcium levels ([Ca2+]i) and in the induction of oxidative burst were measured. 5-VTPA elevated dose-dependently [Ca2+]i and induced the production of reactive oxygen metabolites in leukocytes. 5-VTPA also amplified FMLP-induced increase in [Ca2+]i, but was without an effect on FMLP-induced oxidative burst. On the contrary, 5-VTPA amplified dose-dependently PMA- and DiC8-induced respiratory burst. The present results indicate that 5-VTPA may interfere with transmembrane signalling in human leukocytes. 5-VTPA may elevate [Ca2+]i by acting directly on the membrane, or by acting through Ca(2+)-mobilizing receptors. Moreover, 5-VTPA also clearly amplified responses produced through protein kinase C stimulation. Thus, 5-VTPA may act on human leukocytes by affecting Ca(2+)-metabolism and the activity of protein kinase C.


Subject(s)
Calcium/metabolism , Leukocytes/drug effects , Reactive Oxygen Species/metabolism , Thiazoles/toxicity , Cell Membrane/drug effects , Centrifugation, Density Gradient , Diglycerides/toxicity , Dose-Response Relationship, Drug , Drug Synergism , Humans , Leukocytes/cytology , Leukocytes/metabolism , Luminescent Measurements , N-Formylmethionine Leucyl-Phenylalanine/toxicity , Protein Kinase C/metabolism , Respiratory Burst/drug effects , Tetradecanoylphorbol Acetate/toxicity , Thiazolidines
18.
Br J Pharmacol ; 115(2): 227-36, 1995 May.
Article in English | MEDLINE | ID: mdl-7670725

ABSTRACT

1. Although recent observations suggest that endothelin-1 (ET-1) may play a role in the pathogenesis of asthma, to date little is known about the effects of ET-1 on parameters other than bronchoconstriction. The objectives of the present experiments were to study whether intravenously administered ET-1 could exert pro-inflammatory actions in the guinea-pig lung and to assess the involvement of endothelin ETA and ETB receptors in these events by using the ETA receptor-selective antagonist, FR 139317, the novel ETA/ETB receptor antagonist, bosentan and the ETB receptor-selective agonist, IRL 1620. 2. Bolus i.v. injection of ET-1 (0.1-1 nmol kg-1) to anaesthetized guinea-pigs evoked dose-dependent increases in mean arterial blood pressure which lasted for 6-12 min. This was accompanied by a dose-dependent haemoconcentration (8-15% plasma volume losses) and increases (up to 546%) in albumin extravasation in the trachea, upper and lower bronchi, but not in the pulmonary parenchyma. Qualitatively similar changes were observed following i.v. injection of the ETB receptor agonist, IRL 1620 (0.3 and 1 nmol kg-1), although IRL 1620 appeared to be about 3 times less potent than ET-1. The ETA receptor-selective antagonist, FR 139317 (2.5 mg kg-1) inhibited the ET-1 (1 nmol kg-1)-induced pressor response, haemoconcentration and albumin extravasation by 75, 77 and 60-70%, respectively, whereas it did not attenuate IRL 1620 (1 nmol kg-1)-induced changes. The ETA/ETB receptor antagonist, bosentan (10 mg kg-1) almost completely inhibited the pressor, haemoconcentration and permeability effects of both ET-1 and IRL 1620. 3. ET-1, but not IRL 1620 (0.1-1 nmol kg-1), produced a dose-dependent neutropenia with relative lymphocytosis and monocytosis, but did not induce influx of neutrophil granulocytes into pulmonary tissues or the bronchoalveolar space. ET-1 (1 nmol kg-1)-induced neutropenia was prevented by pretreatment of the animals with FR 139317 (2.5 mg kg-1), bosentan (10 mg kg-1) or adrenaline (90 nmol kg-1), indicating that ET-1 caused intravascular sequestration of neutrophil granulocytes. 4. ET-1 or IRL 1620 (10(-10)-10(-6) M) alone did not activate alveolar macrophages in vitro, whereas at a concentration of 10(-8) M, ET-1, but not IRL 1620, markedly potentiated superoxide production in response to f-Met-Leu-Phe (10(-9)-10(-7) M) and platelet-activating factor (PAF, 10(-9)-10(-7) M), but not to phorbol 12-myristate 13-acetate (10(-9) M). ET-1 did not affect f-Met-Leu-Phe- or PAF-induced increases in intracellular free calcium concentration. This potentiating effect of ET-1 was abolished by FR 139317(1.5 X 10-7 M).5. We conclude that, in addition to evoking airway contractions, ET-1 exerts pro-inflammatory actions via activation of the ETA and to a lesser extent the ETB receptors, and therefore, might contribute to the airway inflammation present in asthma. These findings also suggest the therapeutic potential of ETA/ETB receptor and perhaps ETA receptor-selective antagonists in this disease.


Subject(s)
Endothelins/toxicity , Lung/drug effects , Receptors, Endothelin/metabolism , Animals , Azepines/administration & dosage , Azepines/toxicity , Blood Pressure/drug effects , Bosentan , Bronchopulmonary Sequestration/chemically induced , Capillary Permeability/drug effects , Dose-Response Relationship, Drug , Endothelin Receptor Antagonists , Endothelins/administration & dosage , Guinea Pigs , In Vitro Techniques , Indoles/administration & dosage , Indoles/toxicity , Injections, Intravenous , Lung/metabolism , Macrophages, Alveolar/drug effects , Macrophages, Alveolar/pathology , Male , N-Formylmethionine Leucyl-Phenylalanine/analogs & derivatives , N-Formylmethionine Leucyl-Phenylalanine/toxicity , Neutropenia/chemically induced , Neutrophils/drug effects , Neutrophils/pathology , Peptide Fragments/administration & dosage , Peptide Fragments/toxicity , Plasma Volume/drug effects , Platelet Activating Factor/toxicity , Receptor, Endothelin A , Receptor, Endothelin B , Sulfonamides/administration & dosage , Sulfonamides/toxicity , Superoxides/metabolism , Tetradecanoylphorbol Acetate/toxicity
19.
Blood ; 85(9): 2553-8, 1995 May 01.
Article in English | MEDLINE | ID: mdl-7727783

ABSTRACT

Although our understanding of the molecular interactions that mediate the adhesion of leukocytes to venular endothelial cells has greatly expanded, very little is known about the mechanisms that mediate the passage of leukocytes across the vessel wall in vivo. The aim of the present study was to investigate the role of endogenously formed platelet-activating factor (PAF) in the process of leukocyte extravasation induced by interleukin-1 (IL-1). To determine at which stage of emigration PAF was involved, we studied the behavior of leukocytes within rat mesenteric microvessels by intravital microscopy. Rats were injected intraperitoneally with saline, recombinant rat IL-1 beta (IL-1 beta), or the peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) 4 hours before the exteriorization of the mesenteric tissue. In animals treated with IL-1 beta there was a significant increase in the number of rolling and adherent leukocytes within venules (20- to 40-micron diameter) and in the number of extravasated leukocytes in the tissue. Pretreatment of rats with the PAF receptor antagonist UK-74,505 had no effect on the leukocyte responses of rolling and adhesion, but significantly inhibited the migration of the leukocytes across the vessel wall induced by IL-1 beta (76% inhibition). A structurally unrelated PAF antagonist, WEB-2170, produced the same effect (64% inhibition). However, in contrast, UK-74,505 had no effect on the leukocyte extravasation induced by FMLP, indicating selectivity for the response elicited by certain mediators. These results provide the first line of direct evidence for the involvement of endogenously formed PAF in the process of leukocyte extravasation induced by IL-1 in vivo.


Subject(s)
Chemotaxis, Leukocyte/drug effects , Interleukin-1/pharmacology , Mesentery/blood supply , Platelet Activating Factor/physiology , Receptors, Cell Surface , Receptors, G-Protein-Coupled , Animals , Azepines/pharmacology , Cell Adhesion/drug effects , Dihydropyridines/pharmacology , Edema/chemically induced , Imidazoles/pharmacology , Inflammation , Interleukin-1/toxicity , Leukocytes/drug effects , Leukocytes/physiology , Male , Microcirculation , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , N-Formylmethionine Leucyl-Phenylalanine/toxicity , Platelet Membrane Glycoproteins/antagonists & inhibitors , Rats , Rats, Sprague-Dawley , Recombinant Proteins/pharmacology , Skin/drug effects , Skin/pathology , Triazoles/pharmacology
20.
J Nutr Sci Vitaminol (Tokyo) ; 38(2): 197-201, 1992 Apr.
Article in English | MEDLINE | ID: mdl-1324298

ABSTRACT

(1) We investigated the trophic effect of pectin on the intestinal mucosa injured by formyl methionyl-leucyl-phenylalanine (FMLP), a chemoattractant produced by the intestinal bacterial flora. (2) We first demonstrated that oral administration of FMLP for 7 days reduced the disaccharidase activities and increased the permeability, measured by fluorescein-isothiocyanate-conjugated dextran, of rat small intestine. (3) After 7 days of FMLP administration, rats were divided into fiber-free group which was given liquid elemental diet (Elental) and the pectin group which was given Elental supplemented with 2.5% pectin. (4) After 3 days of feeding (Day 3), the maltase activities of the pectin group was significantly greater than that of the fiber-free group and than that of the initial level just after the 1 week administration of FMLP. At Day 7, there was no difference of maltase activity between the two groups. The sucrase activity of the pectin group was also significantly greater than that of fiber-free group at Day 3. (5) Plasma enteroglucagon was significantly increased in the pectin group. We conclude that pectin-supplemented diet promoted the recovery of disaccharidase activities in the FMLP-injured intestinal mucosa which may be mediated by enteroglucagon.


Subject(s)
Dietary Fiber/pharmacology , Intestinal Mucosa/drug effects , N-Formylmethionine Leucyl-Phenylalanine/toxicity , Pectins/pharmacology , Animals , Dietary Fiber/administration & dosage , Intestinal Mucosa/pathology , Male , N-Formylmethionine Leucyl-Phenylalanine/administration & dosage , Pectins/administration & dosage , Rats , Rats, Inbred Strains , Sucrase/analysis , alpha-Glucosidases/analysis
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