RESUMO
Effects of fish-oil (FO) feeding on serum lipids were investigated in a 42-d controlled diet study. Fifteen healthy male college students were assigned to one of three groups: control (0 g FO); 5 g FO, supplying 2 g n - 3 (omega-3) fatty acids (FAs); or 20 g FO, supplying 8 g n - 3 FAs. In an initial 7-d period subjects consumed a basal diet with no FO. Then FO replaced an equivalent amount of margarine for 5 wk. FO feeding significantly (p less than 0.05) decreased the serum n - 6 FAs, linoleic acid, eicosatrienoic acid, and arachidonic acid. A significant increase in the n - 3 FAs, eicosapentaenoic acid and docosahexaenoic acid, was noted in serum, platelet, and neutrophil phospholipids. The 20-g-FO group showed a 30% decrease (p less than 0.01) in triglycerides after 2 wk FO with no further decrease observed. Thus, 20 g FO produced changes in both FA patterns and triglyceride concentrations whereas 5 g FO produced changes in FA patterns only. Neither FO amount resulted in significant changes in total or HDL cholesterol, apolipoprotein A-I, or apolipoprotein B-100.
Assuntos
Óleos de Peixe/farmacologia , Lipídeos/sangue , Adulto , Apolipoproteínas/sangue , Plaquetas/análise , Colesterol/sangue , Ácidos Graxos/sangue , Óleos de Peixe/administração & dosagem , Humanos , Masculino , Neutrófilos/análise , Fosfolipídeos/sangue , Ensaios Clínicos Controlados Aleatórios como Assunto , Triglicerídeos/sangueRESUMO
Cytosol and membrane fractions from human neutrophils, monocytes, lymphocytes and platelets were separated by SDS/PAGE, blotted on to nitrocellulose and assayed for selective binding of phosphatidylserine (PS). Two PS-binding proteins with apparent molecular masses of 115 kDa and 100 kDa were identified in the cytosol of neutrophils, monocytes and lymphocytes. Corresponding bands along with other PS-binding proteins were detected in platelets in both cytosol and membrane fractions. These proteins were also found to bind protein kinase C (PKC) provided that PS was present. The 115 kDa and 100 kDa proteins (PS-p115/110) were partially purified from neutrophils and were used for the study of PS and PKC binding. The binding of PS did not require Ca2+ or Mg2+ and was inhibited by phosphatidic acid, by 1-alkyl-2-acetylphosphocholine and, to a lesser extent, by other lipids. The binding of PKC, however, was strictly PS- and Ca2(+)-dependent and seems to occur secondarily to PS binding.
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Proteínas de Transporte/sangue , Leucócitos/metabolismo , Fosfatidilserinas/sangue , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Colódio/metabolismo , Citosol/análise , Citosol/metabolismo , Eritrócitos/metabolismo , Eritrócitos/ultraestrutura , Humanos , Leucócitos/enzimologia , Leucócitos/ultraestrutura , Neutrófilos/análise , Neutrófilos/metabolismo , Neutrófilos/ultraestrutura , Fosfatidilserinas/isolamento & purificação , Fosfatidilserinas/metabolismo , Ligação Proteica , Proteína Quinase C/sangueRESUMO
The dormant O2(-)-generating oxidase in plasma membranes from unstimulated neutrophils becomes activated in the presence of arachidonate and a multicomponent cytosolic fraction. This process is stimulated by nonhydrolyzable GTP analogues and may involve a pertussis toxin insensitive GTP-binding protein. Our studies were designed to characterize the putative GTP-binding protein, localizing it to either membrane or cytosolic fraction in this system. Exposure of the isolated membrane fraction to guanosine-5'-(3-O-thio)triphosphate (GTP gamma S), with or without arachidonate, had no effect on subsequent NADPH oxidase activation by the cytosolic fraction. Preexposure of the cytosolic fraction to GTP gamma S alone did not enhance activation of the membrane oxidase. However, preexposure of the cytosol to GTP gamma S then arachidonate caused a four-fold enhancement of its ability to activate the membrane oxidase. This enhancement was evident after removal of unbound GTP gamma S and arachidonate, and was not augmented by additional GTP gamma S during membrane activation. A reconstitution assay was developed for cytosolic component(s) responsible for the GTP gamma S effect. Cytosol preincubated with GTP gamma 35S then arachidonate was fractionated by anion exchange chromatography. A single peak of protein-bound GTP gamma 35S was recovered that had reconstitutive activity. Cytosol preincubated with GTP gamma 35S alone was similarly fractionated and the same peak of protein-bound GTP gamma 35S was observed. However, this peak had no reconstitutive activity. We conclude that the GTP-binding protein regulating this cellfree system is located in the cytosolic fraction. The GTP gamma S-liganded form of this protein may be activated or stabilized by arachidonate.
Assuntos
Citosol/análise , Proteínas de Ligação ao GTP/análise , NADH NADPH Oxirredutases/metabolismo , Neutrófilos/análise , Ácido Araquidônico , Ácidos Araquidônicos/farmacologia , Membrana Celular/enzimologia , Ativação Enzimática , Ácidos Graxos/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato) , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/farmacologia , Humanos , NADPH Oxidases , Tionucleotídeos/farmacologiaRESUMO
Increasing interest in receptor-regulated phospholipase C and phospholipase D hydrolysis of cellular phosphatidylcholine motivates the development of a sensitive and simple assay for the water-soluble hydrolytic products of these reactions, phosphocholine and choline respectively. Choline was partially purified from the methanol/water upper phase of a Bligh & Dyer extract by ion-pair extraction using sodium tetraphenylboron, and the mass of choline was determined by a radioenzymic assay using choline kinase and [32P]ATP. After removal of choline from the upper phase, the mass of residual phosphocholine was determined by converting it into choline by using alkaline phosphatase, followed by radioactive phosphorylation. In addition to excellent sensitivity (5 pmol for choline and 10 pmol for phosphocholine), these assays demonstrated little mutual interference (phosphocholine----choline = 0%; choline----phosphocholine = 5%), were extremely reproducible (average S.E.M. of 3.5% for choline and 2.9% for phosphocholine), and were simple to perform with instrumentation typically available in most laboratories. In addition, the ability to apply the extraction technique to the upper phase of Bligh & Dyer extracts permitted simple analysis not only of choline and phosphocholine, but also of phosphatidylcholine and lipid products of phospholipase C and phospholipase D activity (1,2-diacylglycerol and phosphatidic acid respectively) from the same cell or tissue sample.
Assuntos
Colina/análogos & derivados , Colina/análise , Mastócitos/análise , Neutrófilos/análise , Fosforilcolina/análise , Animais , Etanolamina , Etanolaminas/análise , Humanos , Ratos , Serina/análise , Cloreto de Sódio , SolubilidadeRESUMO
Mastoparan, a peptide toxin from wasp venom, stimulates guanine nucleotide binding and hydrolysis by G proteins. To elucidate the site of mastoparan-G protein interaction, we utilized a polyclonal antibody (R16,17) directed against the carboxyl terminus of the Gi alpha subunit to develop a competitive enzyme-linked immunosorbent assay. We investigated the ability of mastoparan to influence R16,17 antibody binding to G protein alpha subunits in a purified preparation of brain Gi and in neutrophil membrane extracts. Mastoparan antagonized the ability of R16,17 to detect G protein alpha subunits with an IC50 of 15 microM in the purified preparation and with an IC50 of 1 microM for the predominant G protein population in membrane extracts. This reduction was not seen when an unrelated peptide or a peptide of similar charge composition to mastoparan was used in place of mastoparan in the assay. Additionally, antibody R16,17 blocked up to 85% of mastoparan-stimulated GTPase activity. Taken together, these data indicate that the interaction of mastoparan with G protein depends in part on the carboxyl terminus of Gi alpha. Pertussis toxin-catalyzed ADP-ribosylation of Gi alpha markedly inhibited mastoparan-stimulated GTPase activity but only slightly attenuated the ability of mastoparan to recognize G protein. These data suggest that ribosylation inhibits mastoparan-induced G protein activation by a mechanism distinct from the ability of mastoparan to physically interact with G protein. Since mastoparan is thought to mimic hormone-liganded receptors, these findings may be applicable to the mechanism of receptor-Gi protein uncoupling that results from ADP-ribosylation of the G protein.
Assuntos
Venenos de Abelha/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Venenos de Vespas/metabolismo , Adenosina Difosfato Ribose/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Especificidade de Anticorpos , Ligação Competitiva , Química Encefálica , Bovinos , Membrana Celular/análise , Ensaio de Imunoadsorção Enzimática , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Neutrófilos/análise , Peptídeos , Toxina Pertussis , Fatores de Virulência de Bordetella/metabolismo , Venenos de Vespas/farmacologiaRESUMO
A description is given of the methodology, and problems encountered, for the use of a new fluorescent Ca2(+)-indicator dye, fluo-3, in neutrophils and platelets. The higher Kd and longer excitation wavelength of fluo-3 can have significant advantages over fura-2. Although neutrophils and platelets are used as examples, these observations will be applicable to other cell types. The Kd of fluo-3 for binding Ca2+ at 37 degrees C was measured and found to be 864 nM; the previously published value was 400 nM at 22 degrees C. The Kd of fluo-3, like that of fura-2, is therefore very temperature-dependent. Protocols for loading cells, and preventing leakage of fluo-3, are described; probenecid, known to inhibit fura-2 leakage from cells, was found to be essential to get good fluo-3 signals from platelets. Calibration of fluo-3 fluorescence signals to [Ca2+] and methods for obtaining maximum and minimum fluorescence signals are described; these methods differ from those used with fura-2. Agonist-stimulated responses of fluo-3-loaded neutrophils and platelets are shown, and the calculated cytosolic [Ca2+] is comparable with that previously obtained with fura-2. Responses of cells in the presence of plasma are also shown; such measurements, unobtainable with quin2, fura-2 or indo-1, are possible with fluo-3, owing to its longer excitation wavelengths. Co-loading of cells with bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid and fluo-3 is included as an example of how cytosolic [Ca2+] can be buffered and manipulated. Many of these observations will be of value when using fluo-3 (or other Ca2(+)-indicator dyes) in most cell types.
Assuntos
Compostos de Anilina , Plaquetas/análise , Cálcio/sangue , Citosol/análise , Corantes Fluorescentes , Neutrófilos/análise , Xantenos , Benzofuranos , Sangue , Plaquetas/ultraestrutura , Soluções Tampão , Digitonina/farmacologia , Ácido Egtázico/farmacologia , Fura-2 , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/ultraestrutura , Probenecid/farmacologia , Espectrometria de Fluorescência , Trombina/farmacologiaRESUMO
The density (Bmax) and affinity (KD) of beta 2-adrenoceptors in polymorphonuclear leukocytes (PMN) were measured in 29 children and juveniles with mild or moderate asthma and in 25 healthy control subjects using the highly specific radioligand (+/-)-125I-cyanopindolol. No significant difference in Bmax was found between asthmatic subjects without medication and the control group. The asthmatic subjects were divided into four different groups by their actual medication: no medication, adrenergics, theophylline, adrenergics plus theophylline. Analysis of variance revealed highly significant differences between these groups for Bmax (P = 0.0016) but not for KD. In asthmatics on adrenergic therapy Bmax was significantly lower (995 +/- 415) than in asthmatics without medication (1660 +/- 521; P = 0.008). In contrast, Bmax in asthmatics on theophylline therapy was significantly higher (2137 +/- 231; P = 0.009) than in the controls. Bmax was average in the group of asthmatics treated with both adrenergics and theophylline (1619 +/- 547). It is concluded that in asthmatic subjects therapy with theophylline increases the density of beta 2-adrenoceptors and partly prevents their down-regulation induced by adrenergic therapy.
Assuntos
Asma/tratamento farmacológico , Neutrófilos/efeitos dos fármacos , Receptores Adrenérgicos beta/efeitos dos fármacos , Teofilina/farmacologia , Adolescente , Adulto , Asma/fisiopatologia , Criança , Pré-Escolar , Regulação para Baixo , Feminino , Humanos , Masculino , Neutrófilos/análise , Receptores Adrenérgicos beta/análise , Teofilina/uso terapêuticoRESUMO
Chemoattractant stimulation of polymorphonuclear leukocytes is associated with a nearly two-fold rise in actin filament content. We examined the role of the actin monomer sequestering protein, profilin, in the regulation of PMN actin filament assembly during chemoattractant stimulation using a Triton extraction method. Poly-L-proline-conjugated Sepharose beads were used to assess the relative concentration of actin bound to profilin with high enough affinity to withstand dilution (profilin-actin complex) and DNase I-conjugated beads to measure the relative concentration of actin in the Triton-soluble fraction not bound to profilin. Actin associated with the Triton-insoluble fraction (F-actin) was also measured. In unstimulated PMN, the relative concentration of actin bound to profilin was maximum. After FMLP stimulation, profilin released actin monomers within 10 s, with the profilin-actin complex concentration reaching a nadir by 40 s and remaining low as long as the cells were exposed to chemoattractant (up to 30 min). If FMLP was dissociated from PMN membrane receptors using t-BOC, actin reassociated with profilin within 20 s. Quantitative analysis of these reactions, however, revealed that profilin release of and rebinding to actin could account for only a small percentage of the total change in F-actin content. Determination of the total profilin and actin concentrations in PMN revealed that the molar ratio of profilin to actin was 1 to 5.2. When purified actin was polymerized in PMN Triton extract containing EGTA, removal of profilin from the extract minimally affected (12% reduction) the high apparent critical concentration at which actin began to assemble. Although profilin released actin at the appropriate time to stimulate actin assembly during exposure to chemoattractants, the concentration of profilin in PMN was insufficient to explain the high unpolymerized actin content in unstimulated PMN and the quantity of actin released from profilin too small to account for the large shifts from unpolymerized to polymerized actin associated with maximal chemoattractant stimulation.
Assuntos
Actinas/metabolismo , Proteínas Contráteis , Proteínas dos Microfilamentos/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/metabolismo , Actinas/análise , Actinas/sangue , Actinas/isolamento & purificação , Extratos Celulares/análise , Humanos , Proteínas dos Microfilamentos/isolamento & purificação , Músculos/análise , Neutrófilos/análise , Neutrófilos/efeitos dos fármacos , ProfilinasRESUMO
Patients with liver disease may be at risk of zinc depletion. We measured polymorphonuclear cell, mononuclear cell, plasma, and erythrocyte zinc values, and erythrocyte carbonic anhydrase activity to assess zinc status in 17 patients with non-alcoholic liver disease (primary biliary cirrhosis and chronic active hepatitis) and 13 patients with alcoholic liver disease. The plasma zinc concentration was reduced in both patient groups and correlated strongly with the plasma albumin concentration. The mean polymorphonuclear cell zinc value in both groups was similar to that of controls but when results were combined and grouped according to hepatic functional reserve, patients with more severe liver damage (grade C) had a lower polymorphonuclear cell zinc value (mean (SD) 0.86 (0.24) nmol/mg protein) than patients with grade A (1.44 (0.43) nmol/mg protein, p less than 0.01) or grade B liver damage (1.08 (0.30) nmol/mg protein, p less than 0.05), or control subjects (1.26 (0.28) nmol/mg protein, p less than 0.001). The polymorphonuclear cell zinc value did not correlate with other indices of zinc status. The mononuclear cell zinc value was normal in all patients and was unrelated to hepatic damage. The erythrocyte zinc value and carbonic anhydrase activity were raised in alcoholic patients only. Since the polymorphonuclear cell zinc concentration is low in human experimental zinc deficiency and also correlates with tissue zinc, we suggest that our results provide evidence of progressive leucocyte zinc depletion in patients with liver disease.
Assuntos
Hepatite Crônica/sangue , Cirrose Hepática Biliar/sangue , Hepatopatias Alcoólicas/sangue , Zinco/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Neutrófilos/análise , Índice de Gravidade de Doença , Zinco/deficiênciaRESUMO
To determine the susceptibility of laminin to proteolytic degradation by inflammatory cells, soluble laminin was incubated with supernatants from phorbol 12-myristate 13-acetate (PMA)-stimulated human neutrophils. The appearance of laminin cleavage fragments was then detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of supernatants with diisopropylfluorophosphate (DFP), anti-human neutrophil elastase (HNE), and anti-human neutrophil cathepsin G (HNCG) IgGs effectively blocked the degradation of laminin. In contrast, treatment of supernatants with EDTA failed to inhibit laminin digestion, indicating that neutrophil metalloproteinases had little laminin-degrading activity. In additional experiments, laminin was incubated with purified HNE and HNCG. Both enzymes extensively cleaved laminin in a dose- and time-dependent manner yielding similar products, but HNE was generally more potent. Immunofluorescence microscopy of cryostat sections of mouse kidney treated with HNE or HNCG also showed widespread loss of laminin epitopes from basement membranes. The proteolytic degradation of laminin by neutrophil elastase and cathepsin G indicates an important role for these enzymes in basement membrane damage during inflammation.
Assuntos
Catepsinas/farmacologia , Laminina/metabolismo , Neutrófilos/enzimologia , Elastase Pancreática/farmacologia , Catepsina G , Catepsinas/análise , Membrana Celular/análise , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Humanos , Immunoblotting , Laminina/análise , Neutrófilos/análise , Serina EndopeptidasesRESUMO
The presence and amount of the anti-inflammatory protein lipocortin 1 was determined in plasma and peripheral blood leucocytes by a highly specific, enzyme-linked immunosorbent assay. Within 120 min of a single intravenous dose of 100 mg hydrocortisone, the intracellular concentrations of lipocortin 1 in peripheral monocytes in 7 of 8 healthy men increased by a median of 225% (range 129-507%) compared with pretreatment levels, and mononuclear cell-surface lipocortin increased by a median of 224% (range 76-483%). Placebo injections had no effect. There was no increase at any time in free plasma or polymorph-associated lipocortin. In 3 of 4 subjects, induction of lipocortin was also observed when whole unseparated blood was incubated in vitro after steroid administration, but cells which had first been isolated and purified were refractory to such induction. Thus rapid changes in the concentration of an active anti-inflammatory protein can occur in man after normal therapeutic doses of hydrocortisone.
Assuntos
Proteínas de Ligação ao Cálcio/biossíntese , Hidrocortisona/farmacologia , Leucócitos/metabolismo , Adulto , Anexinas , Proteínas de Ligação ao Cálcio/sangue , Avaliação de Medicamentos , Ensaio de Imunoadsorção Enzimática , Humanos , Hidrocortisona/administração & dosagem , Hidrocortisona/sangue , Técnicas In Vitro , Injeções Intravenosas , Leucócitos/análise , Leucócitos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Monócitos/análise , Neutrófilos/análise , Fatores de TempoRESUMO
Results from studies of the retention of contrasting mineral dusts inhaled by rats (for periods of up to three months) and the resultant changes in the phagocyte defence system of the deep lung were examined. The dusts used were titanium dioxide (relatively innocuous) and quartz (relatively toxic). The parameters assessed included the accumulation of material in the lung and lymph nodes during chronic exposure and the associated leukocyte response as assessed by broncho-alveolar lavage. The principal findings were that: (a) low level exposure to titanium dioxide produced no measurable inflammation (as indicated by neutrophil recruitment) but higher concentrations (30, 50, and 90 mg/m3) caused the transfer of dust to lymph nodes and first evidence of inflammation; and (b) for quartz, there was a more prominent response and earlier transfer of material to the lymph nodes. The suggested relation between changes in the neutrophil population and dust accumulation is discussed in terms of a quantitative dosimetric model, from which implications for assessing and managing human exposures emerge.
Assuntos
Poeira/efeitos adversos , Pulmão/patologia , Minerais/efeitos adversos , Animais , Líquido da Lavagem Broncoalveolar/patologia , Exposição Ambiental , Humanos , Modelos Biológicos , Neutrófilos/análise , Doenças Profissionais/etiologia , Quartzo/efeitos adversos , Ratos , Ratos Endogâmicos , Fatores de Tempo , Titânio/efeitos adversosRESUMO
Neutrophils are considered fragile cells, easily damaged by improper handling. Assays of neutrophil function frequently require preliminary isolation procedures that may be potentially harmful. The authors did an extensive investigation of the effects of currently used isolation procedures on a broad spectrum of functional assays that included: 1) phagocytosis; 2) bacterial killing by a culture technique and tritiated thymidine labeling; 3) candidacidal activity by methylene blue dye exclusion and differential Giemsa staining; 4) quantitative unstimulated and histochemical stimulated nitroblue tetrazolium dye reduction; 5) chemiluminescence; 6) random locomotion; and 7) chemotaxis by the agarose method. In addition, cells were examined morphologically by electron microscopy, and the granule density was quantitated by morphometric analysis. Isolation techniques included erythrocyte sedimentation, hypotonic lysis, gradient density separation using Ficoll-Hypaque, and counterflow centrifugal elutriation. The purest neutrophil suspensions were obtained by density gradient separation and counterflow centrifugal elutriation with mean neutrophil percentages of greater than or equal to 94%. Regardless of the isolation procedure, neutrophils were similar in all groups (P greater than .05) in the following studies: phagocytosis, nitroblue tetrazolium dye reduction, bacterial killing, candidal killing, inhibition of candidal germ tube formation, and cytoplasmic granulation. Differences were noted in assays of neutrophil migration and chemiluminescence. Neutrophil suspensions isolated by counterflow centrifugal elutriation and Ficoll-Hypaque had the highest scores for random migration and chemotaxis. These differences can be related to the purity of the neutrophil suspension rather than the harmful effects of the isolation procedures. Erythrocyte contamination affected both the slope and the time to peak response in the chemiluminescence assay. Exposure of neutrophils to cold temperatures (0-4 degrees C) for 1.5 hours impaired both random locomotion and chemotaxis. Current recommendations for the storage, transport, and preparation of leukocytes for neutrophil function studies need to be reassessed.
Assuntos
Separação Celular/métodos , Neutrófilos/fisiologia , Atividade Bactericida do Sangue , Sedimentação Sanguínea , Quimiotaxia de Leucócito/fisiologia , Humanos , Medições Luminescentes , Ativação Linfocitária/fisiologia , Microscopia Eletrônica , Neutrófilos/análise , Neutrófilos/ultraestrutura , Nitroazul de Tetrazólio , Fagocitose/fisiologiaRESUMO
It was found that, in former works, the glucocorticoid receptors (GCR) on peripheral mixed leucocytes in patients with Yang-deficiency were decreased. In this work, the mixed leucocytes were further separated into mononuclear (MNL) and polymorphonuclear (PML) leucocytes, and GCR were determined in each part of leucocytes. GCR on MNL and PML in 6 Yang deficient patients were 3473 +/- 413 and 4433 +/- 651 sites/cell respectively, statistically significant from the normal control group (4462 +/- 962 and 5622 +/- 782 sites/cell respectively, P less than 0.05). GCR on MNL, PML and mixed leucocytes in 5 patients were determined simultaneously, and all lowered from the control group. The results were 3369 +/- 370, 4986 +/- 419 and 4524 +/- 852 sites/cell respectively, with the lowest GCR on MNL and highest on PML.
Assuntos
Leucócitos Mononucleares/análise , Medicina Tradicional Chinesa , Neutrófilos/análise , Receptores de Glucocorticoides/sangue , Adulto , Idoso , Feminino , Humanos , Hidrocortisona/sangue , Masculino , Pessoa de Meia-IdadeRESUMO
Intracellular ionized calcium has been strongly implicated in mediating several responses of human neutrophils to stimulation. However, proteins that serve as effectors of these responses have not been well characterized. To identify proteins that might serve as mediators of the effects of Ca2+ in human neutrophils, we isolated proteins that bind to membrane phospholipids in a Ca2(+)-dependent manner. The most abundant of these, a protein of 33 kD, was readily purified to homogeneity, and was found to bind to phosphatidylserine vesicles in the presence of 2 microM ionized Ca2+. In addition, this purified protein promoted Ca2(+)-dependent aggregation of isolated specific granules from human neutrophils, indicating that it might mediate membrane-membrane contact during processes such as phagosome-lysosome fusion or degranulation. This protein was localized to the cytoplasm of unstimulated neutrophils and found to account for approximately 1% of the cytosol protein. Amino acid sequence of several peptides derived from the purified protein revealed that it is identical to lipocortin III, a recently described member of the annexin family that is scarce in other cells and tissues. The abundance of this protein, together with its Ca2(+)-dependent membrane effects, suggest that it mediates membrane-localized events in stimulated neutrophils, such as phagosome-lysosome fusion or degranulation.
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Proteínas de Ligação ao Cálcio/isolamento & purificação , Cálcio/fisiologia , Grânulos Citoplasmáticos/efeitos dos fármacos , Neutrófilos/análise , Sequência de Aminoácidos , Anexinas , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/fisiologia , Agregação Celular/efeitos dos fármacos , Citosol/análise , Dados de Sequência Molecular , Fosfolipídeos/metabolismoRESUMO
Lipocortins are structurally related, glucocorticoid-inducible proteins that inhibit phospholipase A2 (PLA2), thereby reducing the liberation of arachidonic acid from phospholipids and so limiting the synthesis of eicosanoid inflammatory mediators. This study is the first demonstration of one lipocortin, lipocortin 1 (Lc 1; 37 kDa), in human lung lavage supernatants. In lavage fluid from healthy volunteers, a higher percentage (greater than 70%) of the detected Lc 1 was in its native form, compared to that from patients with abnormal lungs. In patients' lavage fluids, Lc 1 was more likely to be partially degraded (34 kDa). In abnormal bronchoalveolar lavage fluid (BALF), the more polymorphonuclear neutrophils (PMN)/lavage, the lower the proportion of Lc 1 in the native (37 kDa) form (n = 7 pairs, rs = -0.8214, p less than 0.05). Furthermore, when BALF cells were cultured and the harvested conditioned media incubated with pure human recombinant Lc 1, degradation of the 37 kDa form increased with the percentage of PMN (n = 10 pairs, s = -0.7200 after 1 hr; n = 6 pairs, rs = -0.9241 after 6 hr). These results suggest that factors released from the PMN are responsible for Lc 1 degradation in man. When recombinant human Lc 1 was incubated with human neutrophil elastase, the enzyme degraded Lc 1 in a dose-dependent way, suggesting that neutrophil elastase may be one such factor. Since PMNs are ubiquitous at sites of inflammation, it is possible that Lc 1 degradation is a permissive mechanism, which ensures that sufficient inflammation occurs to destroy the provocative stimulus. However, it is equally possible that, in some circumstances, the mechanism may be pathological and that the inactivation of Lc 1 leads to chronic, uncontrolled inflammation.
Assuntos
Líquido da Lavagem Broncoalveolar/análise , Proteínas de Ligação ao Cálcio/análise , Fosfolipases/antagonistas & inibidores , Pneumonia/patologia , Adulto , Idoso , Anexinas , Western Blotting , Líquido da Lavagem Broncoalveolar/citologia , Proteínas de Ligação ao Cálcio/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/análise , Neutrófilos/fisiologia , Pneumonia/diagnóstico , Pneumonia/fisiopatologiaRESUMO
When heat-killed Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS) were injected into mice, liver cell necrosis with infiltrating neutrophils and macrophages were induced. Proliferating cells in the spleen were histochemically investigated. P. acnes injection rapidly produced hyperplasia of neutrophils and macrophages in the red pulp of the spleen. The proportion of Bromodeoxyuridine positive cells reached a peak at 5 days after injection of P. acnes. These results indicate that proliferating cells in the spleen after injection of P. acnes are mainly neutrophils and macrophages, which are the same kinds of infiltrating cells into the liver after injection of P. acnes and LPS.
Assuntos
Fígado/patologia , Baço/patologia , Animais , Bromodesoxiuridina , Divisão Celular , Histocitoquímica , Macrófagos/análise , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Necrose , Neutrófilos/análise , Neutrófilos/patologia , Baço/análiseRESUMO
The antigen which has alpha 2-globulin mobility was identified immunochemically in leukolysate and pus extract. This antigen was localized in polymorphonuclear leukocytes by the immunofluorescence technique in the blood of healthy donors. The alpha 2-globulin of granulocytes (alpha 2GG) is not identical to lactoferrin, lysozyme, granulocyte elastase, fibronectin, fetal hemoglobin, amyloid P-component of the serum. The molecular mass of alpha 2GG is equal to 50 +/- 8 Kd. in the pus extract. The biological role of alpha 2GG is unknown.
Assuntos
alfa-Globulinas/análise , Neutrófilos/análise , Eletroforese das Proteínas Sanguíneas , Imunofluorescência , Humanos , Imunodifusão , Imunoeletroforese , Peso Molecular , Neutrófilos/imunologiaRESUMO
Exposure of murine and human macrophages and human umbilical vein endothelial cells to micromolar concentrations of five microtubule (MT)-depolymerizing agents (colchicine, nocodazole, podophyllotoxin, vincristine, and vinblastine) resulted in a loss of binding sites for iodinated TNF-alpha. The reduction amounted to 40-60% by 1 h and approximately 75% by 2-4 h. In 1 h, specific binding was reduced 50% by 0.1-5 microM of these drugs at 37 degrees C, but not at 4 degrees C. Inactive isomers of colchicine were ineffective, as were microfilament-destabilizing cytochalasins. The active agents did not compete with TNF-alpha R for binding. Antiserum against TNF-alpha did not neutralize the effect of colchicine and nocodazole. PGE1 and dibutyryl-cAMP could not mimic, and cyclooxygenase inhibitors could not prevent the drug effects. All the binding sites were regenerated within 3 h after removal of nocodazole, which binds tubulin reversibly, whereas little recovery was found even 18 h after the removal of colchicine, which binds tubulin irreversibly. These findings suggested that MT disassembly was responsible for the observed downregulation of TNF-alpha R. The protein synthesis inhibitor cycloheximide inhibited binding of TNF-alpha to a similar extent and with a similar time course as colchicine in the absence of added ligand. Neither drug affected binding of IFN-gamma to macrophages, nor binding of TNF-alpha to human polymorphonuclear leukocytes. Thus, an intact MT network appears to be important in maintenance of the steady state of TNF-alpha R on those cells in which TNF-alpha R turns over rapidly in the absence of ligand. The antiinflammatory actions of MT-depolymerizing agents may result in part from their interference with the ability of such cells to respond to TNF-alpha.