RESUMO
Preautoimmune New Zealand Black/White (NZB/NZW) mice immunized with Escherichia coli (EC) double standard (ds) DNA produce antibodies that bind mammalian dsDNA and display specificities similar to spontaneous lupus anti-DNA. Since calf thymus (CT) dsDNA fails to induce these antibodies, these results suggest a special potency of foreign DNA in inducing serological manifestations of lupus in a susceptible host. To assess the effects of DNA immunization on clinical manifestations in NZB/NZW mice, we measured renal disease and survival of mice immunized with either (a) EC dsDNA as complexes with methylated bovine serum albumin (mBSA) in adjuvant; (b) CT dsDNA with mBSA in adjuvant; (c)mBSA alone in adjuvant; or (d) unimmunized. After immunization with EC dsDNA, NZB/NZW mice developed significant levels of anti-dsDNA antibodies. Nevertheless, these mice had less proteinuria, nitrate/nitrite excretion, and glomerular pathology than mice immunized with either mBSA alone, CT dsDNA/mBSA complexes, or unimmunized mice. Survival of the EC dsDNA immunized mice was significantly increased compared with the other mice. Furthermore, immunization of mice after the onset of anti-DNA production and proteinuria stabilized nephritis and prolonged survival. The improvement in renal disease occurred despite the expression of autoantibodies that bound mammalian dsDNA as well as glomerular antigens. These results suggest that bacterial DNA has immunological properties that attenuate murine lupus despite the induction of pathogenic antibodies.
Assuntos
DNA Bacteriano/uso terapêutico , Imunização , Nefrite Lúpica/prevenção & controle , Animais , Escherichia coli , Imunoglobulina G/sangue , Glomérulos Renais/patologia , Nefrite Lúpica/imunologia , Camundongos , Camundongos Endogâmicos NZB , Camundongos Endogâmicos , Nitratos/urina , Nitritos/urina , ProteinúriaRESUMO
The beta 2 integrins (LFA-1, Mac-1, and p150,95) are critical for many adhesive functions of leukocytes. Although the binding of the IgG-opsonized particles occurs normally in the absence of beta 2 integrins, phagocytosis of IgG-opsonized particles by activated neutrophils (PMN) requires these integrins. This observation suggests a role for beta 2 integrins in phagocytosis subsequent to particle binding. To investigate the mechanism of involvement of beta 2 integrins in IgG-mediated functions, we examined the role of beta 2 integrins in adhesion to immune complex (IC)-coated surfaces. Initial adhesion and spreading on IC-coated surfaces were equivalent in control and beta 2-deficient phagocytes. However, both genetically beta 2-deficient PMN and PMN treated with the anti-beta 2 mAb IB4 subsequently detached from the IC-coated surfaces. To determine whether biochemical consequences of IgG activation were also affected by beta 2 deficiency, LTB4 production in response to Fc receptor ligation was assessed. LTB4 production by beta 2-deficient PMN adherent to IC-coated surfaces was markedly decreased in comparison with control PMN. Importantly, LTB4 production by PMN stimulated with fluid phase heat-aggregated IgG also required the beta 2 integrins, showing that the defect was not a simple consequence of abnormal adhesion. In contrast, superoxide production by IC-adherent PMN was equivalent in control and beta 2-deficient PMN. The initial rises in intracytoplasmic [Ca2+]i in response to aggregated IgG also were unaffected by inhibition of beta 2 integrins. These data show that lack of beta 2 integrins does not inhibit all FcR-dependent signal transduction. Finally, LTB4 production by normal PMN adherent to ICs was inhibited by antibodies to FcRII, but not FcRIII, showing that FcRII ligation was required for this effect. Together these data identify a role for the beta 2 integrins in a signal transduction pathway leading to sustained adhesion and LTB4 production in response to IC. Since both beta 2 integrins and FcRII are required for these effects, the data further suggest cooperation between these receptors in generating PMN activation in response to IC stimulation.
Assuntos
Complexo Antígeno-Anticorpo , Imunoglobulina G/farmacologia , Leucotrieno B4/sangue , Antígeno-1 Associado à Função Linfocitária/fisiologia , Neutrófilos/fisiologia , Actinas/análise , Actinas/sangue , Anticorpos Monoclonais , Cálcio/sangue , Adesão Celular , Linhagem Celular , Humanos , Cinética , Leucotrieno B4/biossíntese , Antígeno-1 Associado à Função Linfocitária/genética , Muramidase/sangue , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Receptores de IgG/fisiologiaRESUMO
A central hypothesis in transplantation biology is that resident leukocytes expressing class II histocompatibility antigens may determine the immunogenicity of an organ. By means of a novel method to deplete the kidney of resident leukocytes, essential fatty acid deficiency (EFAD), this hypothesis was tested in an intact, vascular organ. Kidneys subjected to EFAD and thus depleted of resident Ia-positive macrophages survived and functioned when transplanted across a major histocompatibility antigen barrier in the absence of immunosuppression of the recipient. Control allografts were rejected promptly. Allografts from donors subjected to EFAD normalized their lipid composition and were repopulated with host macrophages by 5 days. Administration of Ia-positive cells at the time of transplantation established that the resident leukocyte depletion induced by EFAD was responsible for the protective effect. These observations may provide insights into the mechanisms underlying tissue immunogenicity and the population of normal tissues with resident leukocytes.
Assuntos
Ácidos Graxos Essenciais/fisiologia , Rejeição de Enxerto , Transplante de Rim , Animais , Rim/fisiologia , Fígado/análise , Macrófagos/fisiologia , Fosfolipídeos/análise , Ratos , Ratos Endogâmicos BUF , Ratos Endogâmicos Lew , Transplante HomólogoRESUMO
Essential fatty acid (EFA) deficiency exerts a beneficial effect on immune-mediated glomerulonephritis, preventing both the tissue injury and consequent mortality. Because both macrophages and eicosanoids are thought to play pathogenic roles in glomerulonephritis, and because macrophages play an important role in modulating arachidonate metabolism at sites of renal injury, the effects of EFA deficiency on the population of resident glomerular macrophages and on glomerular eicosanoid generation were examined. EFA deficiency led to a striking reduction in the number of resident glomerular macrophages and a corresponding reduction in the number of resident glomerular Ia+ cells. This phenomenon was not strain-specific, was not due to a decrease in circulating monocytes, was not a function of changes in cell surface labeling characteristics, and was not restricted to a specific subset of glomeruli. In addition, EFA deficiency affected other areas of the renal cortex: a comparable depletion of interstitial macrophages and Ia+ cells was also observed. In conjunction with the decrease in glomerular macrophages seen with the deficiency state, a marked decrease in both basal and angiotensin II-stimulated glomerular eicosanoid production was noted. In contrast to angiotensin II, platelet-activating factor-induced eicosanoid production was not significantly affected by the deficiency state. These changes in glomerular eicosanoid production could not be attributed to changes in glomerular cyclooxygenase or reacylation capacity. Dietary (n-6) fatty acid supplementation, but not (n-3) fatty acid supplementation, reversed both the decrease in glomerular macrophages and the diminished eicosanoid metabolism seen with the deficiency state. Understanding the mechanisms behind the changes in the glomerular microenvironment induced by EFA deficiency may provide a basis for elucidating the protective effect of dietary fatty acid manipulation on immune-mediated glomerulonephritis.
Assuntos
Angiotensina II/antagonistas & inibidores , Ácidos Eicosanoicos/biossíntese , Ácidos Graxos Essenciais/deficiência , Glomérulos Renais/citologia , Macrófagos/citologia , Animais , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Dinoprostona , Ácidos Graxos/análise , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Fígado/análise , Microscopia de Fluorescência , Prostaglandinas E/metabolismo , Ratos , Ratos Endogâmicos Lew , Ratos EndogâmicosRESUMO
Nephrotoxic nephritis (NTN) is characterized by a marked increase in glomerular eicosanoid synthesis, which appears to play an important role in the pathophysiology of this disease model. In this study, we investigated the biochemical and cellular basis of this metabolic change. By examining the enzymatic conversion of exogenous substrates by intact glomeruli, we found that cyclooxygenase, TX synthase, and 5-lipoxygenase activities increased 4-, 8-, and 100-fold, respectively, in acute NTN. PGH2-PGE2 isomerase and leukotriene A4 hydrolase activities did not change. The cellular basis of these changes was examined using dissociated glomerular cells in vitro and by depleting platelets in vivo. Dissociated glomerular cells from nephritic glomeruli (largely mesangial cells and leukocytes) exhibited an enhanced arachidonate metabolism similar to intact nephritic glomeruli. Depletion of neutrophils (PMNs) from these cell preparations by 90% commensurately decreased 5-lipoxygenase and cyclooxygenase activity but had little effect on TX synthase activity. The recovered PMN fraction, however, did exhibit TX synthase activity. Immunocytochemical analysis of dissociated cells using an antiplatelet antibody demonstrated the presence of platelets, both adherent to cells and noncell associated. Depletion of platelets in vivo using this antibody substantially attenuated the increase in glomerular eicosanoid synthesis that accompanied NTN. Platelet depletion also decreased the influx of PMNs into the glomerulus by 50%. These data show that PMNs and platelets colocalize to the glomerulus in acute NTN and are coordinately essential to the increase in glomerular arachidonate metabolism.
Assuntos
Ácidos Araquidônicos/metabolismo , Plaquetas/metabolismo , Glomerulonefrite/metabolismo , Oxirredutases Intramoleculares , Glomérulos Renais/enzimologia , Neutrófilos/metabolismo , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Modelos Animais de Doenças , Epóxido Hidrolases/metabolismo , Glomerulonefrite/sangue , Glomerulonefrite/enzimologia , Soros Imunes , Isomerases/metabolismo , Prostaglandina-E Sintases , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Ratos Endogâmicos Lew , Valores de Referência , Tromboxano-A Sintase/metabolismoRESUMO
Leukotriene (LT) B4 is an important pro-inflammatory autocoid. In order to investigate the potential role of this eicosanoid in renal inflammation, in this study we determined the capability of glomeruli to synthesize this mediator. Glomeruli were able to synthesize LTB4 when provided with exogenous substrate in a dose-dependent fashion in the presence of ionophore A23187. Ionophore, although by itself a weak agonist for LTB4 formation, was required for LTB4 production from exogenous arachidonate. The identity of LTB4 was confirmed by specific radioimmunoassay, high pressure liquid chromatography, and gas chromatography/mass spectrometry. The synthesis of LTB4 was inhibited by BW755C (a lipoxygenase/cyclooxygenase inhibitor) but not indomethacin. Essential fatty acid (EFA) deficiency, obtained by the deprivation of (n-6) fatty acids, is known to exert a protective effect in renal inflammatory states. This dietary manipulation markedly attenuated the ability of glomeruli to synthesize LTB4. In contrast, the synthesis of cyclooxygenase products from exogenous arachidonate was increased by EFA deficiency. Because EFA deficiency has been shown to deplete glomeruli of resident mesangial macrophages, it was hypothesized that this effect accounted for the diminished LTB4 synthesis. To test this hypothesis, glomeruli were depleted of macrophages using x-irradiation. Glomeruli from these animals exhibited a marked decrease in LTB4 synthesis. Glomerular synthesis of cyclooxygenase products was unaffected by irradiation. In sum, glomeruli have the capability to synthesize LTB4, and this capacity is correlated with the presence of glomerular macrophages. EFA deficiency attenuates the ability of glomeruli to synthesize LTB4 by depleting them of macrophages.
Assuntos
Ácidos Graxos/deficiência , Glomérulos Renais/metabolismo , Leucotrieno B4/biossíntese , 4,5-Di-Hidro-1-(3-(Trifluormetil)Fenil)-1H-Pirazol-3-Amina , Animais , Calcimicina/farmacologia , Cromatografia Líquida de Alta Pressão , Dinoprostona/farmacologia , Relação Dose-Resposta a Droga , Indometacina/farmacologia , Camundongos , Pirazóis/farmacologia , Radioimunoensaio , Ratos , Ratos Endogâmicos LewRESUMO
The administration of the aminonucleoside of puromycin (PAN) to rats causes the nephrotic syndrome that is associated with an acute decline in renal function, and an interstitial infiltrate. We examined whether essential fatty acid deficiency (EFAD), which inhibits macrophage infiltration in glomerulonephritis, affects PAN-induced renal dysfunction. Both control and EFAD rats developed proteinuria that resolved over 28 d. After PAN administration, there was a prominent infiltration of macrophages in rats fed a normal diet. The infiltrate was prevented by the EFAD diet. The absence of a macrophage interstitial infiltrate was associated with a significantly higher Cin in the EFAD rats than in controls at 7 d (5.21 +/- 1.19 versus 0.39 +/- 0.08, P less than 0.002 ml/min/kg BW). In addition, CPAH fell to less than 10 ml/min/kg BW by day 7 in controls, but remained the same as normal in the EFAD. After administration of PAN to control rats, there was no increase in urinary thromboxane excretion or an increase in glomerular thromboxane production. Furthermore, the effect of EFAD could not be mimicked by the administration of a thromboxane synthase inhibitor. Irradiation-induced leukopenia in rats on a normal diet markedly improved glomerular filtration and renal blood flow in acutely nephrotic rats. EFAD prevents the interstitial cellular infiltrate and the renal ischemia associated with experimental nephrosis. The recruitment of mononuclear cells into the kidney following PAN directly contributes to the decline in renal function.
Assuntos
Ácidos Graxos Essenciais/deficiência , Nefropatias/prevenção & controle , Puromicina Aminonucleosídeo/toxicidade , Doença Aguda , Animais , Feminino , Rim/patologia , Nefropatias/induzido quimicamente , Leucopenia/fisiopatologia , Macrófagos/patologia , Metacrilatos/farmacologia , Síndrome Nefrótica/induzido quimicamente , Ratos , Ratos Endogâmicos Lew , Tromboxano B2/urinaRESUMO
The molecular mechanism of volatile anesthetic action remains unknown. Attempts to elucidate this mechanism have been complicated by the absence of models in which changes in neuronal cellular properties can be correlated with changes in whole animal anesthetic effect. In this study we describe a model where diet-induced alterations in rat brain fatty acid composition are correlated with alterations in volatile anesthetic potency. Rats maintained on a fat-free diet showed significant depletion of arachidonic acid (20:4 omega 6; 5,8,11,14-eicosatetraenoic acid) and docosahexaenoic acid (22:6 omega 3; 4,7,10,13,16,19,-docosahexaenoic acid) in brain, and a corresponding increase in Mead acid (20: 3 omega 9; 5,8,11-eicosatrienoic acid). These fat-deprived rats were significantly more sensitive to all volatile anesthetics tested than were age-controlled rats on a normal diet. Parenteral supplementation of the fat-deprived animals with linolenic acid (18: 3 omega 3, 9,12,15-octadecatrienoic acid) completely reconstituted the docosahexaenoic acid content of brain without affecting anesthetic sensitivity. In contrast, supplementation of the fat-deprived rats with linoleic acid (18: omega 6; 9,12-octadecadienoic acid) caused a dramatic decrease in anesthetic sensitivity, but only a small change in whole brain arachidonate content. Further analysis revealed that linoleate supplementation of fat-deprived animals resulted in a preferential normalization of the arachidonate content of brain phosphatidylinositol as compared with other brain phosphoglycerides. These results demonstrate for the first time a correlation between changes in membrane composition and anesthetic effect, and indicate that the precise fatty acid composition (perhaps in specific phospholipids) of brain is important in the mechanism of volatile anesthetic action.
Assuntos
Anestésicos/farmacologia , Encéfalo/fisiologia , Ácidos Graxos/fisiologia , Animais , Gorduras na Dieta/fisiologia , Relação Dose-Resposta a Droga , Ácidos Graxos Essenciais/deficiência , Ácidos Graxos Insaturados/fisiologia , Fosfolipídeos/fisiologia , RatosRESUMO
Chemokines are a family of cytokines whose participation in inflammation in vivo remains to be established. Using the rat model of anti-glomerular basement membrane (GBM) nephritis, we found that mRNA for the chemokine CINC (cytokine-induced neutrophil chemoattractant) was induced in the kidney, and the corresponding protein was elaborated by isolated inflamed glomeruli. Production of CINC by glomeruli was unaffected by complement- or leukocyte-depletion prior to disease induction. Cytokines which induce CINC expression in renal cells (TNF-alpha and IL-1 beta) were also expressed in the kidney during glomerular inflammation. TNF-alpha production, unlike CINC, was complement and leukocyte dependent. In vivo administration of anti-CINC, but not anti-human IL-8, IgG selectively attenuated the influx of PMNs into the glomerulus and commensurately diminished proteinuria. The participation of CINC was not tissue-specific: anti-CINC IgG also diminished the influx of PMNs in dermal immune complex inflammation. In sum, we propose that glomerular immune complex deposition/complement activation leads to cytokine production which results in CINC expression by endogenous glomerular cells. The CINC produced plays a contributory role in the influx of PMNs into the glomerulus in the context of the activation of other inflammatory mediators. These results suggest a potential role for CINC homologues, IL-8 and the GRO family of chemokines, in human immune complex-mediated disease.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Fatores Quimiotáticos/fisiologia , Citocinas/fisiologia , Glomerulonefrite/etiologia , Neutrófilos/fisiologia , Animais , Sequência de Bases , Imunoglobulina G/imunologia , Interleucina-1/biossíntese , Interleucina-8/fisiologia , Dados de Sequência Molecular , Coelhos , Ratos , Ratos Endogâmicos Lew , Fator de Necrose Tumoral alfa/biossínteseRESUMO
We used an ELISA employing extracts of human glomerular basement membrane (GBM) to detect, characterize, and evaluate the clinical significance of glomerular-binding IgG in patients with SLE nephritis. Most patients with SLE nephritis exhibited GBM-binding IgG, although many patients with active nonrenal SLE or symptomatic, drug-induced lupus had similar reactivity, albeit at lower levels. IgG binding to GBM in SLE nephritis patients was decreased by DNase pretreatment of GBM, restored after DNase with nuclear antigens (most notably with nucleosomes), inhibited by exogenous nuclear antigens (particularly nucleosomes), but unaffected by exposure of serum to DNase/high ionic strength. The characteristics of IgG binding to GBM largely paralleled the patients' underlying autoimmune response, which was dominated either by antibodies to DNA/nucleosomes or to nucleosomes alone. Binding of lupus sera to nonrenal extracellular matrix (even with nucleosomes) was not equivalent to GBM. Collagenase pretreatment of GBM variably decreased IgG binding, depending on the level and type of binding. SLE nephritis patients with high levels of GBM-binding IgG exhibited more severe disease clinically, but the same renal histopathology, as patients with lower levels. The level of GBM-binding IgG at presentation did not predict the therapeutic response, but decreased in responders to therapy. In sum, glomerular-binding IgG in lupus nephritis binds to epitopes on chromatin, which adheres to GBM in part via collagen. These autoantibodies appear necessary, but not sufficient, for the development of nephritis, and correlate with clinical rather than histopathologic parameters of disease activity.
Assuntos
Imunoglobulina G/imunologia , Glomérulos Renais/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Autoimunidade , Membrana Basal/química , Membrana Basal/efeitos dos fármacos , Membrana Basal/imunologia , Sítios de Ligação de Anticorpos/efeitos dos fármacos , Cromatina/imunologia , Colagenases/farmacologia , DNA/imunologia , Desoxirribonucleases/farmacologia , Epitopos/imunologia , Matriz Extracelular/imunologia , Glomerulonefrite/sangue , Glomerulonefrite/imunologia , Humanos , Lúpus Eritematoso Sistêmico/sangue , Nucleossomos/genética , Nucleossomos/imunologia , Extratos de Tecidos/imunologiaRESUMO
The metabolic and functional alterations which occur during the acute phase of nephrotoxic nephritis (NTN) in rats, a model of immune-mediated glomerulonephritis, result from a cooperative interaction between PMNs and platelets (PLTs). In consequence, we hypothesized that fibrinogen (Fg) might play a critical role in this process and, accordingly, we found that defibrination of animals decreased both the acute phase proteinuria in NTN (approximately 70%) as well as the influx of PLTs and PMNs into the glomerulus (approximately 40-50%). In contrast, blockade of the PLT Fg receptor, alpha IIb beta 3, with the RGD peptidomimetic SC-49992 decreased proteinuria (approximately 90%) without substantially altering the influx of PMNs or PLTs. Immunocytochemistry showed a marked increase in beta 3 integrin expression in inflamed glomeruli which was prevented either by PMN or PLT depletion before disease induction. FACS and immunocytochemical analysis of glomerular cell dissociates demonstrated that beta 3 integrin expression was predominantly on intraglomerular PLTs. In vitro, activated PLTs stimulated the PMN respiratory burst, an interaction which could be inhibited by Fg receptor blockade. In sum, acute NTN is accompanied by a marked increase in glomerular beta 3 integrin expression predominantly due to the influx of PLTs which localize to the glomerulus in a PMN-dependent fashion. Fg appears to serve a major role as a coactivating stimulus for PLT-PMNs in situ via alpha IIb beta 3, potentially mediating the PMN respiratory burst which contributes to proteinuria. Fg may also play a subsidiary role in PMN/PLT comigration.
Assuntos
Plaquetas/fisiologia , Fibrinogênio/fisiologia , Glomerulonefrite/fisiopatologia , Glomérulos Renais/fisiopatologia , Neutrófilos/fisiologia , Animais , Complexo Antígeno-Anticorpo , Plaquetas/efeitos dos fármacos , Complemento C3/análise , Dipeptídeos/farmacologia , Fibrinogênio/análise , Glomerulonefrite/patologia , Imunoglobulina G/análise , Integrina beta3 , Integrinas/análise , Integrinas/biossíntese , Glomérulos Renais/patologia , Antígenos Comuns de Leucócito/análise , Neutrófilos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Adesividade Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Proteinúria , Ratos , Ratos Endogâmicos LewRESUMO
Essential fatty acid (EFA) deficiency is an important tool in probing the role of arachidonic acid (20:4(n-6] in pathophysiologic processes, but requires stringent and prolonged deprivation of (n-6) fatty acids. The present study investigated whether induction of the delta 9 desaturase, which is responsible for the synthesis of oleate, the precursor of 20:3(n-9) which uniquely accumulates in the deficiency state, might serve to accelerate the biochemical and biological effects of EFA deficiency. By alternately fasting and feeding animals a fat-free diet, it was possible to induce markedly the delta 9 desaturase selectively in liver. This dietary manipulation in consequence led to dramatic and rapid changes in hepatic phospholipid fatty acid composition. Within 2 weeks, 20:3(n-9) to 20:4(n-6) ratios in liver phospholipids were several fold greater than those seen in animals fed a fat-free diet alone. These changes, however, contrasted with those seen in the serum and other tissues. The mol% of 20:3(n-9) in serum was not increased by delta 9 desaturase induction and the 20:3(n-9) to 20:4(n-6) ratio was only modestly increased. The effects of delta 9 desaturase induction were even more attenuated in tissues other than the liver. Desaturase induction led to a doubling in the 20:3(n-9) to 20:4(n-6) ratio in phosphatidylcholine in renal cortex and heart, although the ratio in the other phospholipids was unaffected. The 20:3(n-9) to 20:4(n-6) ratio in peritoneal macrophage phospholipids was unaffected by desaturase induction. Thus, delta 9 desaturase induction greatly augments the synthesis of (n-9) fatty acids within the liver and leads to the rapid and substantial accumulation of the abnormal fatty acid, 20:3(n-9). This markedly augmented synthesis of hepatic 20:3(n-9), however, is not reflected in increased plasma levels of 20:3(n-9), and thus the effects of delta 9 desaturase induction are attenuated in tissues other than the liver. These data underscore the notable ability of the liver to maintain polyunsaturated fatty acid homeostasis.
Assuntos
Ácidos Graxos Dessaturases/biossíntese , Ácidos Graxos Essenciais/deficiência , Fígado/metabolismo , Animais , Ácidos Araquidônicos/sangue , Ácidos Araquidônicos/metabolismo , Dieta , Indução Enzimática , Ácidos Graxos Essenciais/sangue , Ácidos Graxos Insaturados , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Fosfolipídeos/sangue , Fosfolipídeos/metabolismo , Estearoil-CoA DessaturaseRESUMO
We have observed that phospholipase A2 (PLA2) activation and arachidonate (AA) release are essential for monocyte/macrophage adherence and spreading. In this study, we addressed the relationship between AA release and cell adherence/spreading in murine resident peritoneal macrophages, and the roles of specific PLA2S in these processes. The PLA2-specific inhibitors, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL, specific for the Ca(2+)-independent PLA2 (iPLA2)) and methyl arachidonoyl fluorophosphonate (MAFP, specific for the Ca(2+)-dependent phospholipase (cPLA2)) inhibited AA release and cell spreading in a correlated fashion but only modestly decreased cell adherence. Cell spreading was normalized by the addition of AA to PLA2-inhibited cells. AA release during spreading was also inhibited by Ca2+ depletion or protein kinase C (PKC) inhibition, and was accompanied by increased (but transient) phosphorylation of cPLA2-Inhibition of macrophage spreading, however, only partially inhibited AA release. Moreover, constitutive AA release was seen in fully spread macrophages which was inhibited by BEL, but not MAFP or Ca2+ depletion. BEL also reversed the phenotype of fully spread cells. These data suggest that macrophage spreading requires the release of AA by the iPLA2 (which appears to be constitutively active) and cPLA2 (which appears to be stimulated by adherence/spreading). Maintenance of macrophage spreading, in contrast, appears to be principally dependent on the iPLA2.
Assuntos
Ácido Araquidônico/metabolismo , Macrófagos Peritoneais/fisiologia , Fosfolipases A/metabolismo , Animais , Ácidos Araquidônicos/farmacologia , Cálcio/metabolismo , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Citosol/enzimologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Cinética , Macrófagos Peritoneais/citologia , Camundongos , Organofosfonatos , Fenótipo , Fosfolipases A/antagonistas & inibidores , Fosfolipases A2 , Proteína Quinase C/metabolismoRESUMO
Our recent findings indicate that glucose-induced insulin secretion from isolated pancreatic islets is temporally associated with accumulation of substantial amounts of free arachidonic acid and that arachidonate may serve as a second messenger for intracellular calcium mobilization in islets. In an effort to determine the source of this released arachidonate, the endogenous fatty acid composition of phospholipids from islets has been determined by thin-layer chromatographic separation of the phospholipids, methanolysis to the fatty acid methyl esters, and quantitative gas chromatographic analyses. The relative abundance of phospholipids in islets as judged by their fatty acid content was phosphatidylcholine (PC), 0.63; phosphatidylethanolamine (PE), 0.23; phosphatidylinositol (PI), 0.067; phosphatidylserine (PS), 0.049. Arachidonate constituted 17% of the total islet fatty acid content, and PC contained 43% of total islet arachidonate. Islets incubated with [3H]arachidonate in the presence of 28 mM D-glucose incorporated radiolabel into PC with a considerably higher specific activity than that of PE, PS or PI. The total fatty acid content of PC from islets incubated with 28 mM glucose for 30 min was significantly lower than that of islets incubated with 3 mM glucose, and smaller effects were observed with PE, PS and PI. The molar decrement in PC arachidonate was 3.2 pmol/islet under these conditions, which is sufficient to account for the previously observed accumulation of free arachidonate (2 pmol/islet). A sensitive method involving negative ion-chemical ionization-mass spectrometric analyses of the pentafluorobenzyl esters of fatty acids derived from trace amounts of lysophosphatidylcholine (lyso-PC) was developed, and glucose-stimulation was found to reduce islet lyso-PC content by about 10-fold. These findings indicate that the insulin secretagogue D-glucose induces phospholipid hydrolysis in islets and suggest that PC may be the major source of free arachidonate which accumulates in glucose-stimulated islets.
Assuntos
Ácidos Araquidônicos/metabolismo , Ácidos Graxos/metabolismo , Glucose/farmacologia , Ilhotas Pancreáticas/metabolismo , Fosfolipídeos/metabolismo , Animais , Células Cultivadas , Hidrólise , Técnicas In Vitro , Ilhotas Pancreáticas/efeitos dos fármacos , Cinética , Masculino , Ratos , Ratos EndogâmicosRESUMO
Inhibition of arachidonate release down-regulates immunoglobulin G-mediated phagocytosis. This arachidonate requirement is selective for IgG-opsonized targets, suggesting that arachidonate may act as a second messenger for Fc gamma receptor-mediated phagocytosis. Here we report the characterization of a phospholipase, activated during phagocytosis, that releases arachidonate from phosphatidylethanolamine in the absence of intracellular calcium ([Ca]i < or = 2 nM). In vitro, a phospholipase with these characteristics was detected in soluble and particulate fractions of human monocyte homogenates. (E)-6-(Bromomethylene)tetrahydro-3-(1-naphthalenyl)2H-pyran-2-one, a drug that selectively inhibits Ca-independent phospholipase A2s, is shown to inhibit IgG-mediated phagocytosis and its associated arachidonate release in intact monocytes as well as the in vitro enzyme activity. These findings provide a link between the whole-cell and in vitro data and present the initial characterization of a receptor-activated, calcium-independent phospholipase from human monocytes.
Assuntos
Cálcio/fisiologia , Imunoglobulina G/fisiologia , Fagocitose/fisiologia , Fosfatidiletanolaminas/fisiologia , Fosfolipases/fisiologia , Ácidos Araquidônicos/metabolismo , Membrana Celular/enzimologia , Citosol/enzimologia , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/metabolismo , Monócitos/química , Monócitos/citologia , Monócitos/fisiologia , Naftalenos/farmacologia , Fosfatidiletanolaminas/metabolismo , Fosfolipases/análise , Fosfolipases A/antagonistas & inibidores , Pironas/farmacologia , Receptores de IgG/análise , Receptores de IgG/metabolismo , Receptores de IgG/fisiologiaRESUMO
Essential fatty acid deficiency (EFAD) is a useful model for studying the role of (n-6) fatty acid metabolism in normal physiology. Because cutaneous manifestations are among the earliest signs of EFAD and because abnormalities in the distribution and function of tissue macrophages have been documented in EFAD rodents, we studied the distribution and function of Class II MHC (Ia) antigen-bearing cells in EFAD C57B1/6 mouse epidermis. Immunofluorescence studies revealed 1.9-9.6 (mean +/- SEM = 5.2 +/- 2.6) times more class II MHC (Ia) antigen-bearing epidermal cells in suspensions prepared from EFAD as compared to normal skin. Analysis of epidermal sheets demonstrated similar numbers of dendritic Ia+ and NLDC145+ cells in EFAD and normal epidermis, however. This discrepancy occurred because some keratinocytes in EFAD epidermal sheets expressed class II MHC (Ia) antigens, whereas keratinocytes in normal mouse epidermis did not. Two-color flow cytometry confirmed that all Ia+ cells in normal epidermis are Langerhans (Ia+ NLDC145+) cells, whereas Ia+ cells in EFAD epidermis are comprised of Langerhans cells and a subpopulation of keratinocytes (Ia+ NLDC145-). Similar levels of Ia antigens were expressed on EFAD and normal Langerhans cells. EFAD and normal epidermal cells were also compared in several in vitro assays of accessory cell function. Epidermal cells prepared from EFAD C57B1/6 mice present the protein antigen DNP-Ova to primed helper T cells more effectively than epidermal cells prepared from normal animals. EFAD epidermal cells are also more potent stimulators of T cells in primary and secondary allogeneic mixed lymphocyte-epidermal cell reactions than normal epidermal cells. The functional differences between EFAD and normal epidermal cells do not appear to result from increased cytokine release or decreased prostaglandin production by EFAD epidermal cells. In view of these findings and the observation that the antigen-presenting cell activity of EFAD epidermal cells correlates with the number of Ia+ keratinocytes in epidermal cell preparations, Ia+ keratinocytes (in the presence of Langerhans cells) may potentiate cutaneous immune responses in vitro and perhaps in vivo as well. These results also suggest that (n-6) fatty acids or metabolites of (n-6) fatty acids are involved in regulating the expression of class II MHC (Ia) antigens by keratinocytes in vivo.
Assuntos
Células Apresentadoras de Antígenos , Epiderme/imunologia , Ácidos Graxos Essenciais/deficiência , Antígenos de Histocompatibilidade Classe II/fisiologia , Queratinócitos/fisiologia , Células de Langerhans/fisiologia , Animais , Células Cultivadas , Citocinas/biossíntese , Indometacina/farmacologia , Inflamação/imunologia , Interleucina-1/farmacologia , Fígado/metabolismo , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Prostaglandinas/biossínteseRESUMO
Nonsteroidal anti-inflammatory drugs (NSAIDs) reduce pain and inflammation by inhibiting the synthesis of prostanoids. However, these drugs inhibit both cyclooxygenase-1 (COX-1), which is essential for the regulation of homeostasis in many tissues, as well as COX-2, which is an important mediator of pain and inflammation. Disruption of COX-1 enzymatic activity by NSAIDs leads to such side effects as interference with platelet functions and gastric ulcers. The recent development of COX-2-specific inhibitors, such as celecoxib, raises the possibility of relieving pain and inflammation with reduced risk of gastrointestinal complications. In Phase II and III studies, celecoxib has demonstrated efficacy in alleviating dental pain and the signs and symptoms of osteoarthritis and rheumatoid arthritis. This COX-2-specific inhibitor was also associated with a markedly lower rate of gastroduodenal injury than is seen typically with NSAIDs. Incidence of most adverse events (including gastrointestinal) and withdrawal rates resulting from adverse events with celecoxib were similar to placebo. Celecoxib appears to be both safe and effective in the treatment of osteoarthritis and rheumatoid arthritis. Its COX-2-specific inhibitory properties thus introduce the possibility of effective relief of arthritic and other types of pain and inflammation with less risk of the mechanism-based toxicities observed with conventional NSAIDs.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Artrite/tratamento farmacológico , Inibidores de Ciclo-Oxigenase/farmacologia , Sulfonamidas/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Celecoxib , Ensaios Clínicos como Assunto , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Humanos , Isoenzimas/efeitos dos fármacos , Proteínas de Membrana , Osteoartrite/tratamento farmacológico , Medição da Dor , Dor Pós-Operatória/tratamento farmacológico , Dor Pós-Operatória/etiologia , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Pirazóis , Sulfonamidas/efeitos adversos , Resultado do TratamentoRESUMO
Rats with essential fatty acid deficiency (EFAD) exhibit mild body growth retardation, diminished leukocyte influx in certain models of inflammation, and skin lesions characterized by ulceration, thinning and decreased pigmentation. In the present study we examined the role of EFAD in cutaneous wound healing, a process in which the inflammatory response and the macrophage play a central role. We reproduced the EFAD condition in Lewis rats (n = 35), and examined its effects in wound healing using the paired rat surgical incision model. Rats were compared with weight-matched controls, receiving standard chow diet. Skin samples harvested at days 5, 7, 14 and 21 post-wounding were evaluated for tensiometry and histology. EFAD rats exhibited all the characteristics of this condition, and the typical alteration of liver lipids. Skin samples harvested at different days post-wounding did not show difference in maximal breaking strength when compared to weight-matched controls. Histological evaluation of skin samples showed no difference in the cellular inflammatory infiltration in either EFAD rats or in weight-matched controls. Immunohistochemical studies revealed no difference in the influx of macrophages in the different groups of rats. Fatty acid supplementation of EFAD rats (n = 7), successfully reversed the EFAD state as assessed by the macroscopic skin and liver changes and liver fatty acid content, without modifying either tensile strength or cellular inflammatory infiltration. Our results suggest that EFAD does not alter the normal course of the cutaneous wound repair in rats, despite all the cutaneous alterations produced by this condition. We conclude that essential fatty acids (EFAs) are not essential for cutaneous wound repair.
Assuntos
Ácidos Graxos Essenciais/fisiologia , Cicatrização/fisiologia , Animais , Peso Corporal , Gorduras Insaturadas na Dieta/administração & dosagem , Ácidos Graxos Essenciais/administração & dosagem , Ácidos Graxos Essenciais/deficiência , Inflamação/patologia , Ácido Linoleico , Ácidos Linoleicos/administração & dosagem , Macrófagos/patologia , Masculino , Ratos , Ratos Endogâmicos Lew , Pele/lesões , Pele/patologia , Pele/fisiopatologia , Resistência à TraçãoRESUMO
This study was conducted to compare the effect of etodolac, nabumetone, and oxaprozin use on gastrointestinal (GI) safety and associated costs based on insurance claims information from practice settings. Data were obtained from a national claims database (MarketScan) for the years 1992 to 1994. The claims data of interest were for patients with arthritis who had used etodolac, nabumetone, or oxaprozin exclusively during a 9-month follow-up period (ONLY groups), or these drugs plus (PLUS groups) the other nonsteroidal anti-inflammatory drugs (NSAIDs) ibuprofen, naproxen, diclofenac, sulindac, piroxicam, ketoprofen, or indomethacin. For each group, we obtained information on the use of inpatient and outpatient services for GI-related events and the associated costs. All GI admissions were classified as NSAID-induced or possibly NSAID-induced events based on International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9 CM) codes. All outpatient upper GI ulcers or bleeding episodes were also identified by specific ICD-9 CM code. There were no significant between-group demographic differences. The proportions of patients with NSAID-induced and possibly NSAID-induced GI admissions were 0.1% and 0.4% for the etodolac-ONLY, 0.3% and 1.0% for the nabumetone-ONLY, and 0.1% and 0.5% for the oxaprozin-ONLY groups, respectively (P > 0.05), and a similar pattern was observed among the PLUS groups. In outpatient settings, 3.9%, 4.2%, and 4.9% of the etodolac-, nabumetone-, and oxaprozin-ONLY patients, respectively (P > 0.05), and 6.0%, 5.3%, and 4.7% of the etodolac-, nabumetone-, and oxaprozin-PLUS patients, respectively, had at least one upper GI ulcer/bleeding claim (P > 0.05). The total health care costs for 9 months were approximately $3000 each for the etodolac-, nabumetone-, and oxaprozin-ONLY groups. Oxaprozin, nabumetone, and etodolac had similar GI-safety and associated-costs profiles based on information from practice settings. Also, in patients who used multiple NSAIDs, the groups did not differ in their GI-safety and cost profiles.
Assuntos
Anti-Inflamatórios não Esteroides/economia , Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite/tratamento farmacológico , Artrite/economia , Butanonas/economia , Butanonas/uso terapêutico , Etodolac/economia , Etodolac/uso terapêutico , Propionatos/economia , Propionatos/uso terapêutico , Adulto , Idoso , Anti-Inflamatórios não Esteroides/efeitos adversos , Butanonas/efeitos adversos , Custos e Análise de Custo , Interpretação Estatística de Dados , Bases de Dados Factuais , Etodolac/efeitos adversos , Feminino , Humanos , Seguro de Serviços Farmacêuticos/economia , Masculino , Pessoa de Meia-Idade , Nabumetona , Oxaprozina , Propionatos/efeitos adversosRESUMO
Osteoarthritis (OA) is responsible for more disability of the lower extremities in the elderly than any other disease in the US. The pain associated with OA is the primary symptom leading to disability in these patients. Current ACR guidelines recommend consideration of acetaminophen for mild-to-moderate pain and conventional non-steroidal anti-inflammatory drugs (NSAIDs) or COX-2 specific inhibitors for moderate-to-severe OA symptoms. The aim of this study was to compare the efficacy and safety of the COX-1 sparing, COX-2 specific inhibitor, celecoxib, with the conventional NSAID naproxen, and placebo, in the treatment of OA of the hip. In this multicenter, randomized, placebo-controlled trial, 1061 patients with symptomatic OA of the hip were randomized to receive celecoxib at doses of 100 mg, 200 mg, or 400 mg/day; naproxen 1000 mg/day; or placebo, for 12 weeks. Patients were evaluated using standard measures of efficacy at baseline, 2-4 days after discontinuing previous NSAID or analgesic therapy, and after 2, 6, and 12 weeks of treatment. All doses of celecoxib and naproxen significantly improved the symptoms of OA, at all time points compared with placebo. This sustained treatment effect of celecoxib was dose dependent. In terms of pain relief and improvement in functional capacity, celecoxib 200 mg/day and 400 mg/day were similarly efficacious and were comparable to naproxen. Both drugs were generally well tolerated. Celecoxib at a dose of 200 mg/day is as effective as a standard therapeutic dose of the conventional NSAID, naproxen, in reducing the pain associated with OA of the hip.