RESUMO
L-selectin, a lectin-like receptor on all leukocyte classes, functions in adhesive and signaling roles in the recruitment of myeloid cells from the blood to sites of inflammation. Here, we consider L-selectin as a determinant of neurological recovery in a murine model of spinal cord injury (SCI). Spinal cord-injured, L-selectin knock-out (KO) mice (male) showed improved long-term recovery with greater white matter sparing relative to wild-type (WT) mice and reduced oxidative stress in the injured cord at 72 h post-SCI. There was a partial and transient reduction in accumulation of neutrophils in the injured spinal cords of KOs at 24 h post-injury. To complement these findings with KO mice, we sought a pharmacologic means for lowering L-selectin levels. We found that diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), induced the shedding of L-selectin from the cell surface of myeloid subsets, specifically neutrophils and non-classical monocytes, in the blood and the injured spinal cord. Diclofenac administration to injured WT mice enhanced neurological recovery to a level comparable to that of KOs but did not improve recovery in KOs. While diclofenac treatment had no effect on myeloid cell accumulation, there was a reduction in oxidative stress at 72 h post-SCI. These findings implicate L-selectin in secondary pathogenesis beyond a role in leukocyte recruitment and raise the possibility of repurposing diclofenac for the treatment of SCI.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Diclofenaco/farmacologia , Inflamação , Selectina L/metabolismo , Leucócitos/metabolismo , Células Mieloides/metabolismo , Estresse Oxidativo/fisiologia , Traumatismos da Medula Espinal , Animais , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/metabolismo , Selectina L/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/imunologia , Traumatismos da Medula Espinal/metabolismoRESUMO
The objective of this research was to examine the effects of dexamethasone (DEX) treatment on various aspects of immunity following administration of a multivalent respiratory vaccine, using a model intended to mimic acute versus chronic stress. Angus × Hereford steers ( = 32; 209 ± 8 kg) were stratified by BW and randomly assigned to 1 of 3 treatments: 1) acute stress (ACU), in which 0.5 mg/kg BW DEX was intravenously administered at 1000 h only on d 0; 2) chronic stress (CHR), in which 0.5 mg/kg BW DEX was intravenously administered at 1000 h on d -3 to 0; or 3) control (CON), in which no DEX was administered. Steers were fitted with indwelling jugular catheters and rectal temperature (RT) recording devices on d -4 relative to vaccination and placed in individual stanchions in an environmentally controlled facility. Blood samples were collected and serum was isolated at -74, -50, and -26 h; at 0.5-h intervals from -4 to 6 h; and at 12, 24, 36, 48, and 72 h relative to multivalent respiratory vaccination at 1200 h on d 0. Additional blood samples were used to analyze complete blood cell count (CBC) and functional capacities of neutrophils. There was a treatment × time interaction ( < 0.01) for RT such that DEX treatment in CHR and ACU steers decreased RT on d -3 and 0, respectively. A treatment × time interaction ( < 0.01) was observed for total white blood cells (WBC), neutrophils, lymphocytes, and monocytes. Specifically, DEX increased WBC and neutrophils in CHR and ACU steers ( < 0.001) yet decreased lymphocytes in CHR steers ( = 0.02) compared with CON steers. Neutrophil concentration increased rapidly, within 2 h of the DEX infusion, in ACU steers. Monocytes transiently increased ( < 0.001) in response to DEX treatment in CHR and ACU steers. In contrast, eosinophils were greater ( < 0.01) in CON steers than in ACU and CHR steers. A treatment × time interaction ( = 0.004) was observed for interferon-γ, with CON cattle exhibiting greater concentrations than the ACU and CHR cattle at 5 h after vaccination, through d 3. Treatment also influenced ( ≤ 0.001) the expression of L-selectin on the surface of neutrophils. The percentage of neutrophils engaging in phagocytosis and the oxidative burst were suppressed ( ≤ 0.001) among only the CHR steers, whereas the intensity of the oxidative burst was suppressed ( ≤ 0.001) for both ACU and CHR steers. These data suggest that our model induced acute and chronic immunosuppression and defined the acute response to a multivalent vaccine in CON steers.
Assuntos
Anti-Inflamatórios/administração & dosagem , Doenças dos Bovinos/prevenção & controle , Bovinos/fisiologia , Dexametasona/administração & dosagem , Infecções Respiratórias/veterinária , Vacinação/veterinária , Administração Intravenosa , Animais , Contagem de Células Sanguíneas/veterinária , Temperatura Corporal/efeitos dos fármacos , Bovinos/imunologia , Terapia de Imunossupressão/veterinária , Interferon gama/efeitos dos fármacos , Interferon gama/metabolismo , Selectina L/efeitos dos fármacos , Selectina L/metabolismo , Masculino , Neutrófilos/efeitos dos fármacos , Distribuição Aleatória , Infecções Respiratórias/prevenção & controle , Estresse Fisiológico/efeitos dos fármacos , VacinasRESUMO
Injection of regulatory T cells (Tregs) followed by sensitization with 2,4,6-trinitrochlorobenzene induced a transient increase in size and cellularity of skin-draining lymph nodes (LNs) in mice. This led us to hypothesize that Tregs may affect the trafficking of T cells from and to peripheral LNs. Two to three hours after sensitization, we found fewer CD8+ T cells expressing CD62L in LNs compared with untreated controls. Injection of wild-type Tregs prevented this down-regulation of CD62L. In contrast, Tregs devoid of the adenosine triphosphate (ATP)-degrading ecto-enzyme CD39 were unable to do so. As for the mechanism of CD62L regulation, we found that ATP, which is released in skin upon hapten-exposure, is inducing the protease ADAM17 in LN-residing T cells via engagement of P2X7 ATP receptors. ADAM17 cleaves CD62L from the surface of CD8+ T cells, which in turn provide a signal for T cells to leave the LNs. This regulation of CD62L is disturbed by the presence of Tregs, because Tregs remove extracellular ATP from the tissue by activity of CD39 and, therefore, abrogate the shedding of CD62L. Thus, these data indicate that the regulation of ATP turnover by Tregs in skin and LNs is an important modulator for immune responses.
Assuntos
Trifosfato de Adenosina/farmacologia , Antígenos CD/imunologia , Apirase/imunologia , Dermatite Alérgica de Contato/imunologia , Imunização/métodos , Selectina L/imunologia , Linfócitos T Reguladores/imunologia , Animais , Western Blotting , Células Cultivadas , Regulação para Baixo , Células Epidérmicas , Epiderme/efeitos dos fármacos , Epiderme/imunologia , Citometria de Fluxo , Fatores Imunológicos/metabolismo , Selectina L/efeitos dos fármacos , Linfonodos/citologia , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Valores de Referência , Linfócitos T Reguladores/metabolismoRESUMO
Multivalent interactions occur frequently in nature, where they mediate high-affinity interactions between cells, proteins, or molecules. Here, we report on a method to generate multivalent aptamers (Multi-Aptamers) that target L-selectin function using rolling circle amplification (RCA). We find that the L-selectin Multi-Aptamers have increased affinity compared to the monovalent aptamer, are specific to L-selectin, and are capable of inhibiting interactions with endogenous ligands. In addition, the Multi-Aptamers efficiently inhibit L-selectin mediated dynamic adhesion in vitro and homing to secondary lymphoid tissues in vivo. Importantly, our method of generating multivalent materials using RCA avoids many of the challenges associated with current multivalent materials in that the Multi-Aptamers are high affinity, easily produced and modified, and biocompatible. We anticipate that the Multi-Aptamers can serve as a platform technology to modulate diverse cellular processes.
Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Selectina L/efeitos dos fármacos , Humanos , Células Jurkat , Selectina L/metabolismo , Ligação ProteicaRESUMO
Leaves of Ligustrum vulgare (common privet) have been used for treatment of oropharyngeal inflammations or as antirheumatic, diuretic, and hypotensive agents in folk medicine in southern Europe. Taking into account that neutrophils are involved in the inflammation, the aim of the study was to determine the effect of an aqueous extract prepared from leaves of Ligustrum vulgare on neutrophil functions. The extract was characterized by the HPLC-DAD-MSn method. The inhibition of reactive oxygen species production by formyl-met-leu-phenylalanine- or phorbol 12-myristate 13-acetate-stimulated neutrophils was determined using luminol- or lucigenin-dependent chemiluminescence. The effect on myeloperoxidase, metalloproteinase 9, and interleukin 8 production by neutrophils was measured by an enzyme-linked immunosorbent assay. Neutrophil elastase release was established spectrophotometrically. The expression of adhesion molecules on neutrophils was analyzed with flow cytometry. The main compounds detected were flavonoids, phenylpropanoids, hydroxycinnamates, and secoiridoids. The inhibition of oxidative burst by the extract was comparable in both stimuli models (formyl-met-leu-phenylalanine: IC50 = 18.2 ± 4.0 µg/mL; phorbol 12-myristate 13-acetate: IC50 = 19.8 ± 3.0 µg/mL). The extract in the concentration range of 5-50 µg/mL inhibited neutrophil elastase release by 23.9-34.1 % and myeloperoxidase release by 24.2-37.4 %. The inhibitory effect on metalloproteinase 9 and interleukin 8 production was around 20 %. The extract in the highest concentration modulated the expression of L-selectin and ß2 integrin. Our results partly support the traditional use of common privet leaves as an anti-inflammatory agent.
Assuntos
Anti-Inflamatórios/farmacologia , Mediadores da Inflamação/metabolismo , Ligustrum/química , Neutrófilos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Antígenos CD18/efeitos dos fármacos , Antígenos CD18/metabolismo , Cromatografia Líquida de Alta Pressão , Ácidos Cumáricos/química , Ácidos Cumáricos/isolamento & purificação , Flavonoides/química , Flavonoides/isolamento & purificação , Humanos , Concentração Inibidora 50 , Iridoides/química , Iridoides/isolamento & purificação , Selectina L/efeitos dos fármacos , Selectina L/metabolismo , Elastase de Leucócito/efeitos dos fármacos , Elastase de Leucócito/metabolismo , Espectrometria de Massas , Neutrófilos/metabolismo , Peroxidase/efeitos dos fármacos , Peroxidase/metabolismo , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Plantas Medicinais , Propanóis/química , Propanóis/isolamento & purificação , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória/efeitos dos fármacosRESUMO
BACKGROUND AND OBJECTIVE: Inflammatory agents, such as lipopolysaccharide (LPS), in periodontal pockets may promote atherogenesis by activating leukocytes. In our previous study, we developed a microchannel chip to observe the cell adhesion process in a fluid system. The objective of this investigation was to examine the mechanism by which periodontopathic bacterial LPS enhances plaque-like formation on a microchannel chip. MATERIAL AND METHODS: To evaluate the effect of Aggregatibacter actinomycetemcomitans LPS on the expression of adhesion molecules, e.g. intercellular adhesion molecule 1 (ICAM-1), lymphocyte function-associated antigen 1 (LFA-1) and L-selectin, on the surface of murine macrophage RAW264.7 cells, the expression of each adhesion molecule was examined by flow cytometry and western blot analysis. Moreover, a flow test on the microchannel chip involving anti-adhesion molecule antibodies was conducted to clarify which adhesion molecule is related to plaque-like formation of RAW264.7 cells. RESULTS: The expressions of ICAM-1 and LFA-1 on the surface of RAW 264.7 cells increased following 12 h culture with LPS; L-selectin expression was unaffected. An increase in ICAM-1 expression was also confirmed by western blot analysis. The flow test revealed that anti-ICAM-1 antibody inhibited plaque-like formation of LPS-stimulated macrophages on the micropillars of the microchannel chip. CONCLUSION: These findings indicate that ICAM-1 plays an important role in plaque-like formation of LPS-stimulated macrophages. Our microchannel chip is a suitable tool for the investigation of etiological factors of atherosclerosis, including periodontitis, in vitro.
Assuntos
Aggregatibacter actinomycetemcomitans/fisiologia , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Selectina L/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Antígeno-1 Associado à Função Linfocitária/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Animais , Anticorpos , Aterosclerose/patologia , Western Blotting , Adesão Celular/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Técnicas de Cultura de Células , Linhagem Celular , Citometria de Fluxo , Molécula 1 de Adesão Intercelular/análise , Selectina L/análise , Dispositivos Lab-On-A-Chip , Antígeno-1 Associado à Função Linfocitária/análise , CamundongosRESUMO
The D-mannose binding lectin ArtinM from Artocarpus integrifolia, previously known as KM+ and artocarpin, is considered a stimulant of Th1-type immunity, which is able to confer resistance to some intracellular pathogens. In addition, ArtinM induces neutrophil migration by haptotaxis through simultaneous interactions of its carbohydrate recognition domains (CRDs) with glycans expressed on the extracellular matrix and the neutrophil surface. In the present study, we have expanded the characterization of ArtinM as a neutrophil activator. Exposure of neutrophils to ArtinM for 15 min resulted in tyrosine phosphorylation of intracellular proteins, a process that was selectively inhibited by d-mannose or mannotriose. Shortly after stimulation, neutrophils secreted high levels of LTB(4) and underwent shedding of L-selectin from their surface. Exposure to ArtinM enhanced neutrophil functions, such as respiratory burst and zymozan and Listeria monocytogenes phagocytosis. In addition, ArtinM-stimulated neutrophils displayed increased CXCL-8 secretion and TLR2 gene transcription. These results demonstrate that ArtinM is able to induce potent neutrophil activation, a feature that should be strongly considered in the assessment of the lectin capacity to confer resistance against infections.
Assuntos
Movimento Celular , Lectinas de Ligação a Manose/farmacologia , Ativação de Neutrófilo , Neutrófilos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Lectinas de Plantas/farmacologia , Humanos , Interleucina-8/biossíntese , Interleucina-8/imunologia , Interleucina-8/farmacologia , Selectina L/efeitos dos fármacos , Selectina L/imunologia , Selectina L/metabolismo , Leucotrieno B4/biossíntese , Leucotrieno B4/imunologia , Neutrófilos/imunologia , Fagocitose/imunologia , Receptor 2 Toll-Like/efeitos dos fármacos , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismoRESUMO
BACKGROUND: Increased soluble L-selectin levels have been shown to attenuate local inflammation-mediated microvascular leakage, and failure to generate high levels has been associated with increased risk of acute respiratory distress syndrome in septic patients. We hypothesized that failure to shed L-selectin in systemic inflammation would result in increased local inflammation-induced leukocyte adherence and microvascular leakage. METHODS: Using intraperitoneal lipopolysaccharide (LPS) or control bicarbonate buffered saline (BBS) and intrascrotal TNFalpha or BBS, mice were randomized to systemic inflammation (LPSip + BBSis), local inflammation (BBSip + TNFis), both (LPSip + TNFis), or control (BBSip+BBSis). Furthermore, mice received intraperitoneal L-selectin Sheddase inhibitor (Ro31-9790) or control vector. With intravital microscopy on cremaster muscle, we measured leukocyte-endothelial cell interactions and microvascular leakage (permeability index). Surface L-selectin was measured by flow cytometry (MCF). RESULTS: Without Ro31-9790, systemic inflammation attenuated increases induced by local inflammation in leukocyte adherence and vascular leakage. Ro31-9790 significantly increased adherence and leakage in systemic and systemic + local inflammation. L-selectin was shed progressively by increasing degrees of inflammation. Ro31-9790 limited this shedding of L-selectin. CONCLUSION: In systemic inflammation, L-selectin shedding is required to limit local inflammation-mediated leukocyte adherence and microvascular leakage. Failure to shed L-selectin may increase leukocyte-mediated end-organ injury in septic patients.
Assuntos
Permeabilidade Capilar/imunologia , Selectina L/fisiologia , Neutrófilos/fisiologia , Sepse/fisiopatologia , Animais , Adesão Celular , Células Endoteliais/fisiologia , Fluoresceína-5-Isotiocianato , Ácidos Hidroxâmicos/farmacologia , Inflamação/tratamento farmacológico , Selectina L/efeitos dos fármacos , Selectina L/metabolismo , Lipopolissacarídeos , Masculino , Camundongos , Distribuição Aleatória , Escroto/irrigação sanguínea , Sepse/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Leukocyte extravasation is initiated by an interaction of selectin adhesion molecules and appropriate carbohydrate ligands. Targeting those interactions seems a promising approach to treat chronic inflammation. We developed a beta-1, 3-glucan sulfate (PS3) with inhibitory activity toward L and P-selectins under static conditions. Here, detailed investigation showed inhibition of P- and L-selectins, but not E-selectin under flow conditions (relative reduction of interaction with appropriate ligands to 34.4+/-16.6, 8.5+/-3.6, or 99.5+/-9.9%, respectively, by PS3 for P-, L- or E-selectin). Intravital microscopy revealed reduction of leukocyte rolling in skin microvasculature from 22.7+/-5.0 to 12.6+/-4.0% after injection of PS3. In the next experiments, mice were sensitized with 2,4,-dinitrofluorobenzene (DNFB), and lymphocytes were transferred into syngeneic recipients, which were challenged by DNFB. Inflammatory responses were reduced when immunity was generated in mice treated with PS3 or in L-selectin-deficient mice. No effect was observed when L-selectin-deficient donor mice were treated with PS3, further suggesting that PS3 acted primarily through inhibition of L-selectin. Elicitation of a contact hypersensitivity response was reduced in P-selectin-deficient and in PS3-treated mice. Again, PS3 had no effect in P-selectin-deficient mice. PS3 is a potent P- and L-selectin inhibitor that may add to the therapy of inflammatory diseases.
Assuntos
Dermatite de Contato/tratamento farmacológico , Dermatite de Contato/imunologia , Glucanos/farmacologia , Selectina L/efeitos dos fármacos , Selectina-P/efeitos dos fármacos , Transferência Adotiva , Alérgenos/imunologia , Alérgenos/farmacologia , Animais , Comunicação Celular/efeitos dos fármacos , Células Cultivadas , Dermatite de Contato/metabolismo , Dinitrofluorbenzeno/imunologia , Dinitrofluorbenzeno/farmacologia , Modelos Animais de Doenças , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Glucanos/uso terapêutico , Humanos , Selectina L/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Selectina-P/metabolismo , beta-Glucanas/farmacologia , beta-Glucanas/uso terapêuticoRESUMO
Atherogenesis involves the migration of leukocytes into vascular subendothelial space, a process mediated by endothelial and leukocyte cell adhesion molecules. Endothelial molecules are assessed indirectly via serum levels, but leukocyte molecules can be assessed directly. We have therefore hypothesized that leukocyte adhesion molecules are altered to a greater degree in hypercholesterolemia than serum endothelial adhesion molecules. We examined 29 subjects with hypercholesterolemia and 27 controls at baseline and after 12 weeks of atorvastatin treatment (20 mg/day). Expression of leukocyte integrins CD11a, CD11b, CD18, and CD49d and of L-selectin was measured by flow cytometry. Serum ICAM-1, E-selectin and von Willebrand factor were measured by ELISA. Expression of leukocyte adhesion molecules was significantly higher in patients at baseline than in the controls, except for CD11a. Expression significantly decreased after atorvastatin in most adhesion molecules except for CD11b. In contrast, there was no effect of hypercholesterolemia and/or atorvastatin on the serum endothelial molecules. Leukocyte but not endothelial adhesion molecules were influenced by hypercholesterolemia and by lipid lowering treatment. Leukocyte molecules may therefore be a more sensitive marker of atherogenesis than endothelial molecules. Our results support the role of increased leukocyte adhesiveness in atherogenesis.
Assuntos
Anticolesterolemiantes/uso terapêutico , Moléculas de Adesão Celular/efeitos dos fármacos , Ácidos Heptanoicos/uso terapêutico , Hipercolesterolemia/sangue , Integrinas/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Pirróis/uso terapêutico , Adulto , Atorvastatina , Moléculas de Adesão Celular/sangue , Selectina E/sangue , Selectina E/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Humanos , Hipercolesterolemia/tratamento farmacológico , Integrinas/sangue , Molécula 1 de Adesão Intercelular/sangue , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Selectina L/sangue , Selectina L/efeitos dos fármacos , Leucócitos/metabolismo , Masculino , Análise por Pareamento , Pessoa de Meia-Idade , Valores de Referência , Estatísticas não Paramétricas , Fator de von Willebrand/efeitos dos fármacos , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND: Acute alcohol intoxication is associated with increased susceptibility to infection. In host defense, the expression of adhesion molecules such as beta2-integrin and l-selectin on leukocytes is involved in leukocyte migration to inflamed organ tissue. To elucidate the mechanisms underlying the immunosuppressive effects of ethanol, we investigated whether ethanol pretreatment may influence the changes in adhesion molecule expression induced by lipopolysaccharide (LPS) or interleukin (IL)-8 in human whole blood. METHODS: Ethanol was added to samples of human whole blood (final concentration: 0%, 0.2%, 0.4%, and 0.8%). Samples were assigned to an unstimulated group and an LPS-stimulated group. In another set of experiments, stimulation was induced by IL-8. After fluorescence labeling of alphaM-subunit of beta2-integrin (CD11b) and l-selectin (CD62L), the expression of CD11b and CD62L were measured using flow cytometry. RESULTS: Stimulation with LPS significantly upregulated CD11b expression (5.9 +/- 0.9 to 16.3 +/- 1.8, p < 0.05). Ethanol inhibited this LPS-induced upregulation of CD11b (p < 0.001). Stimulation with IL-8 significantly upregulated CD11b expression (5.3 +/- 1.7 to 7.5 +/- 2.7, p < 0.01) and this IL-8-induced upregulation of CD11b was also inhibited by ethanol pretreatment (p < 0.001). In contrast, ethanol did not modify CD62L expression in either unstimulated or stimulated groups. CONCLUSION: The impairment of CD11b expression on leukocytes suggests that alcohol intake interferes with the migration of leukocytes to sites of inflammation, which may explain, in part, why alcohol intoxication increases susceptibility to infection.
Assuntos
Anti-Infecciosos Locais/farmacologia , Antígenos CD18/efeitos dos fármacos , Etanol/farmacologia , Selectina L/efeitos dos fármacos , Leucócitos/metabolismo , Antígenos CD18/sangue , Sobrevivência Celular , Humanos , Técnicas In Vitro , Interleucina-8 , Selectina L/sangue , Lipopolissacarídeos , Masculino , Valores de ReferênciaRESUMO
Selectins mediate tethering and rolling of leukocytes along the endothelium in a shear force-dependent manner. This key step in the cellular immune response is a target for experimental anti-inflammatory therapies. In the present paper we have examined the inhibitory activity of the minimal selectin ligand sialyl Lewis x (SiaLe(x)), its isomer sialyl Lewis a (SiaLe(a)) and sulfated tyrosine (sTyr) residues under dynamic flow reflecting the rheological conditions in the blood stream. The monomeric ligands were compared to multivalent polyacrylamide (PAA)-based conjugates under defined flow conditions on the molecular level, using surface plasmon resonance (SPR) technology, and on the cellular level, using a parallel-plate flow chamber. SPR measurements showed that a spatial arrangement of binding epitopes mimicking the selectin binding motif of the natural ligand PSGL-1 inhibits L-selectin binding successfully with IC(50) values in the nanomolar range. Using a flow chamber adhesion assay it could be shown that the multivalent inhibitors efficiently blocked rolling and tethering of NALM-6 pre-B cells transfected with human L-selectin to activated endothelium and that the inhibitory activity increased with rising shear stress. While PAA-conjugates were almost not inhibitory at low shear stress, NALM-6 cell rolling was nearly completely inhibited at high shear stress. The results indicate that multimeric conjugates of SiaLe(x), SiaLe(a) and sTyr are highly effective inhibitors of L-selectin-mediated cell adhesion particularly under flow conditions. Consequently, SiaLe(x), SiaLe(a) and/or sTyr on macromolecular carriers may be promising candidates for anti-inflammatory therapy.
Assuntos
Gangliosídeos/metabolismo , Selectina L/metabolismo , Resinas Acrílicas , Técnicas Biossensoriais , Antígeno CA-19-9 , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Gangliosídeos/farmacologia , Hemorreologia , Humanos , Selectina L/efeitos dos fármacos , Antígeno Sialil Lewis X , Ressonância de Plasmônio de Superfície , Tirosina/análogos & derivados , Tirosina/metabolismo , Tirosina/farmacologiaRESUMO
OBJECTIVE: Somatostatin regulates immune inflammatory response via apoptosis and adhesion of leukocytes in many diseases. This article reported a study that aimed to observe the mechanism and effect of somatostatin on the immune inflammatory response through apoptosis and adhesion of leukocytes in severe acute pancreatitis. METHODS: Thirty-eight patients with severe acute pancreatitis, that fulfilled the guidelines for the treatment of severe acute pancreatitis of China and Balthazar computed tomography severity index (>or=5) were enrolled consecutively. Nineteen of these patients received our routine treatment and 19 received additional somatostatin. In all patients the expressions of CD4, CD8, CD95/CD95 ligand and CD18/CD62 ligand on leukocytes were determined by flow cytometry, both upon admission and on the fourth day. Thirty healthy volunteers constituted the normal healthy group. RESULTS: In the treatment group, CD4, CD4:CD8 ratio and CD62 ligand on leukocytes increased from 11.4+/-8.2, 0.47+/-0.10 and 25.5+/-9.2 to 22.1+/-9.7, 0.68+/-0.11 and 36.2+/-11.7 (P<0.05) respectively, while CD95 ligand on both lymphocyte and polymorphonuclear cells increased from 0.65+/-0.21 and 0.76+/-0.29 to 1.18+/-0.32 and 1.58+/-0.43 after treatment with somatostatin (P<0.05). Furthermore, lactate dehydrogenase, aspartate aminotransferase, amylase, C reactive protein and acute physiology and chronic healthy evaluation (APACHE II) score in the treatment group reduced faster than those in the control group (P<0.05), though there was no difference in mortality (15.7% vs 5.3%) between the two patient groups (P>0.05). CONCLUSION: Somatostatin can modulate the immune inflammatory response and the severity of severe acute pancreatitis through apoptosis and adhesion of leukocytes, but this modulatory effect by itself is not strong enough to improve the final.
Assuntos
Anti-Inflamatórios/farmacologia , Linfócitos/efeitos dos fármacos , Pancreatite/tratamento farmacológico , Somatostatina/farmacologia , Regulação para Cima/efeitos dos fármacos , APACHE , Doença Aguda , Adulto , Idoso , Apoptose/efeitos dos fármacos , Antígenos CD18/efeitos dos fármacos , Antígenos CD18/metabolismo , Antígenos CD4/efeitos dos fármacos , Antígenos CD4/metabolismo , Antígenos CD8/efeitos dos fármacos , Antígenos CD8/metabolismo , Adesão Celular/efeitos dos fármacos , Feminino , Citometria de Fluxo , Humanos , Selectina L/efeitos dos fármacos , Selectina L/metabolismo , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Pancreatite/imunologia , Estudos Prospectivos , Receptor fas/efeitos dos fármacos , Receptor fas/metabolismoRESUMO
OBJECTIVE: The first step in atherosclerosis is characterized by the adherence of lymphocytes and monocytes to cell adhesion molecules expressed by endothelial cells. The precise mechanism by which steroid hormones may be exerting a protective action against atherogenesis remains unclear. Therefore, we wanted to investigate the effect of tibolone on the circulating levels of various selectins in postmenopausal women. METHODS: Thirty healthy postmenopausal women were enrolled in a prospective, randomized, double blind, placebo-controlled outpatient trial. RESULTS: Patients treated with tibolone revealed a significant decrease for the variables sE-selectin, sL-selectin, and sPECAM-1 after 8 weeks of treatment. CONCLUSIONS: By reducing leukocyte adhesion molecule expression on human endothelial cells, tibolone may have the intrinsic potential to exert additional, lipid-independent, cardiovascular protective effects that may explain the clinical benefits of cardiovascular diseases in postmenopausal women.
Assuntos
Selectina E/efeitos dos fármacos , Moduladores de Receptor Estrogênico/farmacologia , Selectina L/efeitos dos fármacos , Norpregnenos/farmacologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/efeitos dos fármacos , Pós-Menopausa , Idoso , Idoso de 80 Anos ou mais , Método Duplo-Cego , Selectina E/sangue , Moduladores de Receptor Estrogênico/administração & dosagem , Feminino , Humanos , Selectina L/sangue , Fator Estimulador de Colônias de Macrófagos/sangue , Fator Estimulador de Colônias de Macrófagos/efeitos dos fármacos , Pessoa de Meia-Idade , Norpregnenos/administração & dosagem , Selectina-P/sangue , Selectina-P/efeitos dos fármacos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/sangue , Pós-Menopausa/sangue , Pós-Menopausa/efeitos dos fármacos , Estudos Prospectivos , Resultado do TratamentoRESUMO
INTRODUCTION: We synthesized sulfo-glycolipid, beta-SQAG9 (designate square beta-SQAG9 liposome, because it efficiently forms a liposome structure) that possessed immunosuppressive effects such as inhibition of T-cell responses in human allogeneic MLR and skin allograft survival in rats, and bound to CD62L (L-selectin) in vitro. In this study, we further investigated the immunosuppressive mechanism in vivo by beta-SQAG9 liposome in a skin-allografted rat model. METHODS: ACI rats (RT1(a)) were grafted skin of LEW rats (RT1(1)) treated with PBS or beta-SQAG9 liposome IV once a day for 7 days. Subsequently, we investigated the population of T cells and CD62L(+) T-cell subset in the spleen, axillary lymph nodes (ALNs), and peripheral blood of skin-allografted rats by two-color flow cytometry. RESULTS: Five of 11 (45.5%) rats that were treated with 50 mg/kg beta-SQAG9 liposome showed graft survival and another showed moderate rejection in graft. The CD62L(+) T-cell subset population in ALNs of beta-SQAG9 liposome-treated rats decreased in a dose-dependent manner. No significant difference in the T-cell population was observed between the beta-SQAG9 and control groups. These data suggest that beta-SQAG9 could bind to the CD62L(+) T-cell subset in vivo as well as in vitro and affect T-cell migration, which might lead to T-cell tolerance in vivo.
Assuntos
Glicolipídeos/farmacologia , Sobrevivência de Enxerto/imunologia , Imunossupressores/farmacologia , Selectina L/imunologia , Transplante de Pele/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Sobrevivência de Enxerto/efeitos dos fármacos , Selectina L/efeitos dos fármacos , Lipossomos , Modelos Animais , Ratos , Ratos Endogâmicos ACI , Ratos Endogâmicos Lew , Subpopulações de Linfócitos T/efeitos dos fármacos , Linfócitos T/efeitos dos fármacosRESUMO
L-selectin is a leukocyte cell-surface protein that facilitates the rolling of leukocytes along the endothelium, a process that leads to leukocyte migration to a site of infection. Preventing L-selectin-mediated rolling minimizes leukocyte adhesion and extravasation; therefore, compounds that inhibit rolling may act as anti-inflammatory agents. To investigate the potential role of multivalent ligands as rolling inhibitors, compounds termed neoglycopolymers were synthesized that possess key structural features of physiological L-selectin ligands. Sulfated neoglycopolymers substituted with sialyl Lewis x derivatives (3',6-disulfo Lewis x or 6-sulfo sialyl Lewis x) or a sulfatide analog (3,6-disulfo galactose) inhibited L-selectin-mediated rolling of lymphoid cells. Functional analysis of the inhibitory ligands indicates that they also induce proteolytic release of L-selectin. Thus, their inhibitory potency may arise from their ability to induce shedding. Our data indicate that screening for compounds that promote L-selectin release can identify ligands that inhibit rolling.
Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Selectina L/efeitos dos fármacos , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Animais , Anti-Inflamatórios/síntese química , Sequência de Carboidratos , Linhagem Celular , Linhagem Celular Tumoral , Regulação para Baixo , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicoproteínas/farmacologia , Humanos , Selectina L/química , Selectina L/metabolismo , Ligantes , Linfócitos/efeitos dos fármacos , Camundongos , Mimetismo Molecular , Dados de Sequência MolecularRESUMO
We previously reported that synthetic sulfo-glycolipid, 3-O-(6-deoxy-6-sulfono-beta-D-glucopyranosyl)-1,2-di-O-acylglycerol (beta-SQDG(18:0)) which was deduced from sulfonoquinovosyl-diacylglycerols of sea urchin possessed immunosuppressive effects, such as human mixed lymphocyte reaction (MLR) and skin allograft in rat, and that these effects were caused by contact inhibition between T-cells and antigen presenting cells (APCs). Here, we investigated the mechanism of these immunosuppressive effects on human MLR by beta-SQAG9 which had been newly synthesized from beta-SQDG(18:0) to improve structural stability in water solution. CD62L+ T-cells in peripheral blood predominantly respond to APCs, and beta-SQAG9 inhibited the response of CD62L+ T-cell subset in human allogeneic MLR. Surprisingly, it was demonstrated that beta-SQAG9 bound to L- and P-selectin (CD62L and P) molecule in vitro. Meanwhile, beta-SQAG9 efficiently formed liposome structure and bound to L-selectin on the cell surface of CD62L+ T-cell subset but might not be incorporated into the cells. Because the immunosuppressive effects of beta-SQAG9 disappeared when beta-SQAG9 liposome was changed to soluble form by detergent, the liposome structure of beta-SQAG9 was presumed to be essential for these effects. These findings suggested beta-SQAG9 to be a novel drug with a unique immunosuppressive action.
Assuntos
Glicolipídeos/farmacologia , Selectina L/efeitos dos fármacos , Selectina L/imunologia , Linfócitos T/imunologia , Antígenos de Superfície/efeitos dos fármacos , Antígenos de Superfície/imunologia , Glicolipídeos/síntese química , Glicolipídeos/química , Células HL-60 , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Lipossomos , Teste de Cultura Mista de Linfócitos , Selectina-P/efeitos dos fármacos , Selectina-P/imunologia , Ligação Proteica , Linfócitos T/efeitos dos fármacosRESUMO
The etiology and pathogenesis of the majority of diseases that make up the acute leukemias is unknown. A change in IL-1beta and L selectin concentrations is most likely to occur in the course of subtype M2 of acute myeloid leukaemia (AML). The purpose of our study was to examine the change in concentrations of these molecules mentioned above in blood plasma, culture supernatant and isolated, broken granulocytes in AML patients in both exacerbation and remission of the disease and in healthy control group. Cytokine concentration assay was performed by means of ready immunoenzymatic sets of ELISA type. The examinations were carried out in leukaemic leukocyte cultures Neupogen--stimulated or nonstimulated. Mitogen was added to activate granulocytes and to provoke blastic transformation. A significant increase in IL-1beta concentration was found in AML--exacerbation and remission of the disease in blood plasma, culture supernatant and isolated, broken granulocytes. In all cases L-selectin concentrations were increased in exacerbation and decrease in remission of AML after typical chemotherapy in comparison to controls. A significant increase between the concentrations of cytokines were observed in cultures Neupogen--stimulated and non-stimulated.
Assuntos
Antineoplásicos/farmacologia , Interleucina-1/metabolismo , Selectina L/efeitos dos fármacos , Selectina L/metabolismo , Leucemia Mieloide Aguda/metabolismo , Adulto , Estudos de Casos e Controles , Técnicas de Cultura de Células , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , PolôniaRESUMO
OBJECT: We previously reported that erythromycin (EM) affected the adhesion molecules that interact with neutrophils and inhibited neutrophil infiltration in a rat experimental otitis media model. PURPOSE: The purpose of the present study was to examine whether EM affects the adhesion of rat neutrophils to endothelial cells. METHODS: The adhesion of neutrophils to WK-5 endothelial cells was evaluated. One group of rats was given EM for 2 weeks, while the other group received no treatment. The adhesion of peripheral-blood neutrophils and neutrophils accumulated in the middle ear from these two groups were compared. RESULTS: Two-week EM pretreatment inhibited adhesion of neutrophils to WK-5 cells. The adhesion of neutrophils accumulated in the middle ear cavity to WK-5 cells was significantly higher than that of peripheral-blood neutrophils from control rats. CONCLUSION: The results suggest that EM affects the adhesion of neutrophils to endothelial cells.
Assuntos
Antibacterianos/farmacologia , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Eritromicina/farmacologia , Neutrófilos/efeitos dos fármacos , Otite Média/tratamento farmacológico , Animais , Antibacterianos/uso terapêutico , Adesão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Endotélio Vascular/citologia , Eritromicina/uso terapêutico , Selectina L/análise , Selectina L/efeitos dos fármacos , Antígeno de Macrófago 1/análise , Antígeno de Macrófago 1/efeitos dos fármacos , Distribuição Aleatória , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: Cytokines and adhesion molecules have a decisive role in the development of early inflammatory response as well as the late sequelae of sepsis. Because L-selectin-deficient mice are protected from lethal endotoxemia, blockade of L-selectin may provide a useful therapeutic option in human sepsis. Heparin has immunomodulatory properties and effectively inhibits L- and P-selectin binding in vitro. We therefore investigated whether clinically applied doses of unfractionated or low-molecular-weight heparin affect early inflammatory response in human endotoxemia. DESIGN: The study was randomized, double-blinded, placebo-controlled, in three parallel groups consisting of 30 healthy male volunteers. SETTING: University medical center. INTERVENTIONS: All subjects received a 2-ng/kg intravenous bolus of lipopolysaccharide and 10 mins later unfractionated heparin, low-molecular-weight heparin, or placebo as bolus primed continuous infusion for 6 hrs. MEASUREMENTS AND MAIN RESULTS: Lipopolysaccharide infusion induced similar increases of tumor necrosis factor-alpha, interleukin-6, interleukin-8, C-reactive protein, and soluble E-selectin levels in all treatment groups. CD11b expression increased by approximately 400%, but L-selectin decreased by 41% in the placebo arm 6 hrs after lipopolysaccharide infusion. Interestingly, both heparins (in particular unfractionated heparin) decreased L-selectin down-regulation as compared with placebo. Similarly, the decrease in lymphocyte counts was significantly less in the unfractionated heparin group during the first 24 hrs (p <.05 vs. placebo) CONCLUSIONS: Heparins displayed little effects on cytokine production and endothelial cell activation in endotoxemia. Of note, however, unfractionated heparin reduced L-selectin down-regulation and lymphocytopenia. These could present novel mechanisms of action of unfractionated heparin.