RESUMO
OBJECTIVE: To assess the effect of the lipidosterolic extract of Serenoa repens (LSESr) on in vitro cell proliferation in biopsies of human prostate MATERIAL AND METHODS: Cell proliferation was assessed by incorporation of [3H]thymidine followed by historadiography. RESULTS: Basic fibroblast growth factor (b-FGF) induced a considerable increase in human prostate cell proliferation (from +100 to +250%); the glandular epithelium was mainly affected, minimal labeling being recorded in the other regions of the prostate. Similar results were observed with epidermal growth factor (EGF), although the increase in cell proliferation was not recorded in some cases. Lovastatin, an inhibitor of hydroxymethylglutaryl coenzyme A, antagonized both the basal proliferation and the growth factor-stimulated proliferation of human prostate epithelium (EGF, mean inhibition approximately 80-95%; b-FGF, mean inhibition approximately 40-90%). Geraniol, a precursor of both farnesyl pyrophosphate and geranylgeranyl pyrophosphate, and farnesol, the precursor of farnesyl pyrophosphate, increased cell proliferation only in some prostate specimens, this effect being antagonized by lovastatin. LSESr did not affect basal prostate cell proliferation, with the exception of two prostate specimens in which a significant inhibition of basal proliferation was observed with the highest concentration of LSESr (30 micrograms/ ml). In contrast, LSESr inhibited b-FGF-induced proliferation of human prostate cell cultures; this effect was significant for the highest concentration of LSESr (30 micrograms/ml). In some prostate samples, a similar inhibition was also noted with lower concentrations. Unsaturated fatty acids (UFA), in the range 1-30 ng/ml), did not affect the basal prostate cell proliferation, only a slight increase in cell proliferation was noted in 1 prostate specimen. UFA (1, 10 or 30 micrograms/ml) markedly inhibited the b-FGF-induced cell proliferation down to the basal value. Lupenone, hexacosanol and the unsaponified fraction of LSESr markedly inhibited the b-FGF-induced cell proliferation, whereas a minimal effect on basal cell proliferation was noted. CONCLUSIONS: Despite the large variability in the response of the prostate samples to b-FGF, these results indicate that LSESr and its components affect the proliferative response of prostate cells to b-FGF more than their basal proliferation.
Assuntos
Antagonistas de Androgênios/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Extratos Vegetais/farmacologia , Próstata/efeitos dos fármacos , Monoterpenos Acíclicos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Fator de Crescimento Epidérmico/farmacologia , Farneseno Álcool/farmacologia , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Lovastatina/farmacologia , Masculino , Extratos Vegetais/química , Próstata/patologia , Hiperplasia Prostática/patologia , Serenoa , Terpenos/farmacologiaRESUMO
The tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP or Goralatide), a physiological regulator of hematopoiesis, inhibits the entry into the S-phase of murine and human hematopoietic stem cells. It has been shown to reduce the damage to specific compartments in the bone marrow resulting from treatment with chemotherapeutic agents, ionizing radiations, hyperthermy, or phototherapy. The present study was performed to assess the therapeutic potential of AcSDKP in vivo in reducing both the toxicity and the hematopoietic damage induced by fractionated administration of doxorubicin (DOX), a widely used anticancer drug. Here we showed that AcSDKP could reduce DOX-induced mortality in mice and could protect particularly the long-term reconstituting cells (LTRCs) in addition to colony forming units-spleen, high proliferative potential colony-forming cells, and colony-forming units-granulocyte-macrophage (CFU-GM) from DOX toxicity. The protection against DOX-induced mortality in mice was improved when AcSDKP was administered for 3 days, at a dose of 2.4 micrograms/d, by continuous subcutaneous (SC) infusion or fractionated s.c. injections starting 48 hours before DOX treatment. Moreover, the recovery of the CFU-GM population in the AcSDKP-DOX-treated mice was optimized by the subsequent administration of granulocyte colony-stimulating factor (G-CSF). The coadministration of AcSDKP with DOX may improve its therapeutic index by reducing both acute hematotoxicity on late stem cells and progenitors and long-term toxicity on LTRCs. Optimization of these treatments combined with G-CSF may provide an additional approach to facilitate hematopoietic recovery after cancer chemotherapy.
Assuntos
Antibióticos Antineoplásicos/toxicidade , Células da Medula Óssea/efeitos dos fármacos , Doxorrubicina/toxicidade , Inibidores do Crescimento/administração & dosagem , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Oligopeptídeos/administração & dosagem , Animais , Células da Medula Óssea/patologia , Antagonismo de Drogas , Células-Tronco Hematopoéticas/patologia , Humanos , Camundongos , Análise de SobrevidaRESUMO
Although the lipidic extract of Serenoa repens (LESSr, Permixon, Sereprostat) is widely used in patients suffering from benign prostatic hypertrophy (BPH), its mechanism of action is not fully elucidated. It has been demonstrated that infiltration of the prostate by inflammatory cells is one of the aetiologic factors involved in the development of BPH. These inflammatory cell types, such as polymorphonuclear neutrophils (PMNs), produce chemotactic mediators and contribute to the development of the disease. Among the chemotactic factors generated by inflammatory cell types, the derivatives of arachidonic acid have been extensively studied. For instance, leukotriene (LT) B4 is one of the most potent chemotactic factors for PMNs and also exhibits a wide range of biological activities. In order to investigate the potential action of LESSr on arachidonate metabolism, and particularly on the synthesis of LTB4, the effect of this extract on the in vitro synthesis of LT by human PMNs stimulated with the calcium ionophore A23187 was investigated. LESSr significantly inhibits the production of 5-lipoxygenase metabolites (5-HETE, 20-COOH LTB4, LTB4 and 20-OH LTB4) at concentrations as low as 5 microg/ml. Such an effect of LESSr was also observed in the presence of exogenous arachidonic acid (20 microg/ml) and when f-MLP was used as the agonist, suggesting that inhibition of LTB4 production by the extract was unrelated to phospholipase A2 blockade and independent of the stimulating agent. The capability of LESSr to antagonize 5-lipoxygenase metabolites production may contribute, at least partly, to the understanding of its therapeutic activity on the inflammatory component of BPH.
Assuntos
Calcimicina/farmacologia , Ionóforos/farmacologia , Leucotrieno B4/biossíntese , Neutrófilos/metabolismo , Extratos Vegetais/farmacologia , Araquidonato 5-Lipoxigenase/metabolismo , Sobrevivência Celular , Humanos , Masculino , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Hiperplasia Prostática/tratamento farmacológico , SerenoaRESUMO
Retinoids exert various functions including anti-proliferative and anti-inflammatory effects on many cell types including keratinocytes and are widely used in skin diseases, such as psoriasis and acne. We have previously shown that human keratinocytes express low affinity immunoglobulin E receptor (FcepsilonRII/CD23) when stimulated with interleukin-4. Immunoglobulin E ligates CD23 and induces the production of nitrites (reflecting the mobilization of the nitric oxide [NO]-pathway) and tumor necrosis factor-alpha by human keratinocytes. Here, 13-cis and all-trans retinoic acid (RA) were shown to reduce the production of nitrites by immunoglobulin E-activated keratinocytes by 80% in a time- and concentration-dependent fashion. As a consequence, RA derivatives also reduced the production of tumor necrosis factor alpha by these cells by 70%. The level of inducible NO synthase activity in activated human keratinocytes was significantly decreased upon treatment of the cells with RA derivatives (inhibition by 60% of the mean inducible NO synthase activity with 13-cis RA, 2 microM). Treatment for 24 h with RA derivatives almost completely abolished transcription of inducible NO synthase-specific mRNA in activated keratinocytes. Therefore, RA derivatives downregulate tumor necrosis factor-alpha release and the NO-transduction pathway through the inhibition of inducible NO synthase transcription. Together, our data provide evidence for inhibition of the NO-pathway by 13-cis and all-trans retinoic acid on CD23-activated human keratinocytes. These data may clarify the mechanism of the anti-inflammatory activity of RA derivatives in skin diseases.
Assuntos
Queratinócitos/metabolismo , Óxido Nítrico Sintase/metabolismo , Receptores de IgE/fisiologia , Tretinoína/análogos & derivados , Divisão Celular/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Humanos , Nitritos/antagonistas & inibidores , Retinoides/farmacologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
The synthetic polyribonucleotide poly(A).poly(U) induces 2',5'-oligoadenylate synthetase activity in the murine macrophage cell line J774A1. The possible role of several cytokines involved in macrophage activation (i.e., IL-1, IL-6, TNF, and IFN) was examined in the present study. It was first demonstrated that among the anticytokine antibodies, only monoclonal antibodies directed against IL-6 inhibited the induction of 2',5'-oligoadenylate synthetase by poly(A).poly(U) in a dose-dependent manner. Moreover, it was established that poly(A).poly(U) elicited IL-6 production in J774A1 cells in a time-and dose-dependent manner. Consequently, the effect of IL-6 on 2',5'-oligoadenylate synthetase activity was studied. IL-6 either alone or in combination with IL-1 and TNF did not induce 2',5'-oligoadenylate synthetase activity. IL-6 did not potentiate IFN-gamma-induced 2'-5'-oligoadenylate synthetase activity. In contrast, addition of IL-6 to the incubation medium potentiated the stimulation of 2'-5'-oligoadenylate synthetase activity by IFN-alpha. These results suggest that IL-6 is a necessary but not sufficient factor in the induction of 2'-5'-oligoadenylate synthetase activity in the J774A1 cell line by poly(A).poly(U).
Assuntos
2',5'-Oligoadenilato Sintetase/biossíntese , Indutores de Interferon/farmacologia , Interferon-alfa/fisiologia , Interleucina-6/fisiologia , Macrófagos/efeitos dos fármacos , Poli A-U/farmacologia , Animais , Anticorpos/sangue , Interferon-alfa/imunologia , Interleucina-1/imunologia , Interleucina-1/fisiologia , Interleucina-6/imunologia , Macrófagos/metabolismo , Camundongos , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Human basophils activated through high-affinity immunoglobulin E (IgE) receptors (Fc epsilon RI) are involved in the late phase of the allergic reaction. To investigate the possible involvement of protein-tyrosine kinases in this activation we used human acute basophilic leukemia (ABL) cells in culture as well as a pure population of normal basophils in vitro-derived from human bone marrow precursor cells (HBMB). ABL cells were 50-80% basophils at various stages of maturation as assessed by staining, morphology, ultrastructure, and flow cytometry analysis, and only basophils in ABL cells expressed Fc epsilon RI. Aggregation of Fc epsilon RI by IgE and anti-IgE, IgE and antigen, or anti-Fc epsilon RI monoclonal antibodies on ABL cells or on HBMB, led to increased tyrosine phosphorylation of 120-, 100-, 80-, 72-, 50- to 65-, and 38-kDa substrates. Tyrosine phosphorylations in ABL cells were in basophils because 1) they were detected after a 5-s stimulation, 2) they were observed under conditions where mediator release is minimal, i.e., in the absence of extracellular calcium, 3) hapten addition during antigen stimulation resulted in almost total disappearance of tyrosine phosphorylations within 30 s. There was correlation between histamine release and tyrosine phosphorylation in anti-IgE dose-responses and in dose-responses of the tyrosine kinase inhibitor genistein. The tyrosine kinase p72syk was detected in the cells. Stimulation of ABL cells for 1 min resulted in extracellular calcium-independent tyrosine phosphorylation and activation of p72syk. Therefore, tyrosine kinases are involved in the early steps of human Fc epsilon RI signaling in basophils. Tyrosine kinases and their substrates could represent new potential therapeutic targets to prevent the development of the allergic reaction.
Assuntos
Basófilos/imunologia , Precursores Enzimáticos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de IgE/fisiologia , Idoso , Ativação Enzimática , Humanos , Imunofenotipagem , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia Basofílica Aguda , Masculino , Agregação de Receptores , Transdução de Sinais , Quinase Syk , Células Tumorais CultivadasRESUMO
Mouse bone marrow cells cultured for 6 days in the presence of recombinant murine IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were used as a source of precursors responsive to eosinopoietins. They were further cultured for 7 days in the presence of either a combination of recombinant cytokines or supernatants of bone marrow-derived mast cells (BMMC) activated with either immunological or nonimmunological stimuli. Cytosmears of collected cells were analyzed for eosinophil contents and allowed to demonstrate that supernatants of passively sensitized BMMC support both total cell proliferation and eosinophil production, after various periods of incubation with monoclonal rat anti-mouse IgE antibodies (the 6HD5 mAbs). In contrast, a stimulation with 100 ng/ml dinitrophenylated bovine serum albumin (DNP-BSA) did not generate supernatants displaying such bioactivities. Low doses of methyl ester of L (but not D)-leucine or of the calcium ionophore A23187 also allowed the release of eosinopoietic bioactivities. In addition, immunoreactive IL-5, GM-CSF, and IL-3 were quantified in the BMMC supernatants. These results demonstrate that activated BMMC are able to effect eosinophil production.
Assuntos
Eosinófilos/citologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Interleucina-3/fisiologia , Interleucina-5/fisiologia , Mastócitos/imunologia , Animais , Células da Medula Óssea , Divisão Celular , Células Cultivadas , Eosinófilos/imunologia , Hematopoese , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Recombinantes , Fatores de TempoRESUMO
Human keratinocytes (HK) generate nitric oxide (NO) and proinflammatory mediators following activation with either IgE/anti-IgE immune complexes or a combination of lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Recently, interleukin-10 (IL-10) has been shown to down-regulate various inflammatory responses and to be secreted by lymphocytes and dendritic cells during skin inflammatory reactions. We show here that IL-10 down-regulates the production of tumor necrosis factor (TNF)-alpha and IL-6 by activated HK. Also, induction of inducible nitric oxide synthase (iNOS) expression in HK by IgE/anti-IgE or LPS/IFN-gamma is significantly reduced by the addition of IL-10. This effect is dose dependent and correlates with reduction of iNOS mRNA production and enzyme level. Therefore, IL-10 down-regulates NO-mediated HK inflammatory responses and may thus participate in the regulation of the skin immune network.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Imunoglobulina E/farmacologia , Interleucina-10/farmacologia , Interleucina-6/biossíntese , Queratinócitos/efeitos dos fármacos , Óxido Nítrico Sintase/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Anticorpos Anti-Idiotípicos/farmacologia , Sequência de Bases , Células Cultivadas , Depressão Química , Indução Enzimática/efeitos dos fármacos , Humanos , Interferon gama/farmacologia , Interleucina-6/genética , Interleucina-6/metabolismo , Queratinócitos/enzimologia , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Óxido Nítrico/biossíntese , Proteínas Recombinantes , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The effects of two monocyclic monoterpenes, limonene and sobrerol, known as inhibitors of farnesyltransferase activity, were studied on monocrotaline (MCT)-induced lung injury, pulmonary hypertension and right ventricular hypertrophy in male Wistar rats. After 14 days, pulmonary arterial pressure values and the right ventricle to left ventricle plus septum weight ratios, RV/(LV + S), were markedly increased in rats subcutaneously injected with MCT (60 mg/kg). Limonene and sobrerol, administered daily at the oral dose of 400 mg/rat, markedly decreased the MCT-induced alterations. After treatment for 21 days, limonene still prevented pulmonary hypertension and the increase in RV/(LV + S). Both monoterpenes also reduced the increase in pulmonary arterial media thickness, the development of interstitial fibrosis and the increase in the number of macrophages in intra-alveolar spaces and of lymphocytes around the pulmonary veins. The present data indicate that treatment of rats with inhibitors of farnesyltransferase, like limonene and sobrerol, regulate the development of pulmonary hypertension.
Assuntos
Alquil e Aril Transferases , Hipertensão Pulmonar/prevenção & controle , Pulmão/irrigação sanguínea , Monocrotalina/toxicidade , Proteínas Musculares/metabolismo , Neovascularização Patológica/prevenção & controle , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Terpenos/uso terapêutico , Transferases/antagonistas & inibidores , Animais , Cicloexenos , Avaliação Pré-Clínica de Medicamentos , Farnesiltranstransferase , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/fisiopatologia , Hipertrofia Ventricular Direita/induzido quimicamente , Hipertrofia Ventricular Direita/fisiopatologia , Hipertrofia Ventricular Direita/prevenção & controle , Limoneno , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Neovascularização Patológica/induzido quimicamente , Prenilação de Proteína/efeitos dos fármacos , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/patologia , Ratos , Ratos Sprague-Dawley , Terpenos/farmacologiaRESUMO
The present study examined the effect of beta 2-adrenoceptor agonists on the interleukin-4 (IL-4)-driven IgE production and on the possible mechanisms of action of these compounds. We present evidence that salbutamol and fenoterol potentiated the IL-4-induced IgE production by human peripheral blood mononuclear cells (PBMC). No significant effect of incubation in the presence of beta 2-adrenoceptor agonists on IgG, IgA and IgM production was observed. Salbutamol and fenoterol inhibited interferon-(IFN)-gamma production by PHA-activated human PBMC suggesting that the blockade of the production of this cytokine could possibly explain the enhancement of IgE production. Salbutamol and fenoterol potentiated the IL-4-induced production of sCD23 whereas no effect on CD23 expression was observed. The potentiating effect of salbutamol on IgE production was blocked by two antagonists of beta 2-adrenoceptor, namely butoxamine and D,L-propranolol, suggesting a beta-adrenoceptor-mediated event. These results demonstrate that beta 2-adrenoceptor stimulation results in an increase in IgE production by human B lymphocytes.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Imunoglobulina E/biossíntese , Monócitos/metabolismo , Albuterol/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Humanos , Imunoglobulinas/sangue , Interleucina-4/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfocinas/sangue , Receptores Adrenérgicos beta/fisiologia , Receptores de IgE/metabolismo , SolubilidadeRESUMO
The effect of the antagonist of platelet-activating factor (PAF), BN 50730, on PAF-and antigen- induced increase in microvascular leakage, using Evans blue dye as an index of permeability, was investigated in rat pulmonary tissues. PAF (1 microgram/kg, iv) induced a marked increase in Evans blue dye content in trachea, main bronchi and small bronchi, that was significantly reduced upon pretreatment of the rats with BN 50730 (25 mg/kg, orally) and by the serotonin antagonist, methysergide (1 mg/kg, iv), only in the small bronchi. Serotonin also induced an increase in microvascular leakage in the three tissues that was significantly inhibited when the animals were treated with methysergide but not by BN 50730. In contrast, histamine and lyso-PAF did not induce significant increase in Evans blue dye content. Intravenous injection of antigen to IgE-sensitized rats induced a biphasic increase in vascular permeability. An early increase in vascular permeability in trachea, main bronchi and small bronchi was observed 10 minutes after the injection of the antigen, and this phenomenon was significantly reduced upon treatment of the rats with methysergide, whereas, BN 50730 was ineffective. A late increase in vascular permeability was noted in the three tissues, with a maximum at 120 minutes and representing 30-40% of the magnitude of the first phase. Administration of BN 50730 (25 mg/kg) to the animals, evoked a significant inhibition of this increase in microvascular leakage, whereas, methysergide only significantly reduced the one induced by antigen in the trachea.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Azepinas/farmacologia , Permeabilidade Capilar/efeitos dos fármacos , Metisergida/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Antagonistas da Serotonina/farmacologia , Triazóis/farmacologia , Administração Oral , Animais , Antígenos/imunologia , Brônquios/metabolismo , Azul Evans , Extravasamento de Materiais Terapêuticos e Diagnósticos , Imunoglobulina E/imunologia , Injeções Intravenosas , Masculino , Fator de Ativação de Plaquetas/farmacologia , Ratos , Ratos Sprague-Dawley , Serotonina/metabolismo , Organismos Livres de Patógenos Específicos , Tienopiridinas , Traqueia/metabolismoRESUMO
Upon in vitro stimulation with 10 microM ionophore A 23187 for 5 min at 37 degrees C, the generation of leukotriene (LT) C4 in polymorphonuclear leukocytes (PMNL) from nine untreated cystinotic children was significantly increased compared with that in eight control children (p < 0.01) and 25 normal adults (p < 0.001) (417.4 +/- 70.0 versus 177.0 +/- 30.9 and 164.9 +/- 19.5 pmol/l x 10(7) cells, respectively). Concomitantly with the increased generation of LTC4, LTB4 production in PMNL from untreated cystinotic children was decreased compared with controls, whereas the total amount of LTA4 derivatives was similar in the three groups. The increase in LTC4 production was not related to the number of eosinophils present in the PMNL preparations from cystinotic children, which was similar to that of control subjects. PMNL from cystinotic children treated with cysteamine, an aminothiol compound that decreases the intracellular cystine content, generated smaller amounts of LTC4 upon ionophore A 23187 stimulation than PMNL from untreated cystinotic children. In addition, abrogation of the cysteamine treatment for 3 or 4 d led to an increase in LTC4 production. These findings suggest that the metabolic abnormalities taking place in infantile cystinosis may favor the biosynthesis of LTC4 from PMNL.
Assuntos
Cistina/sangue , Cistinose/sangue , Leucotrieno C4/biossíntese , Neutrófilos/metabolismo , Adulto , Calcimicina/farmacologia , Criança , Pré-Escolar , Cistinose/genética , Eosinófilos/fisiologia , Heterozigoto , Humanos , Leucotrieno C4/sangue , Estimulação QuímicaRESUMO
The present study examined the in vitro and in vivo effect of salbutamol on IgE production in the mouse. The present results show that salbutamol potentiates the in vitro interleukin 4 (IL-4)-induced IgE production from lipopolysaccharide-activated murine B lymphocytes. This effect is dose-dependent and is observed at concentrations above 10 nM. In vivo, when ovalbumin (OA)-sensitized BALB/c mice were treated with a daily injection of salbutamol, an increase of the anti-OA IgE levels in the serum was observed as compared to sensitized animals. Such an effect was observed at doses above 1 microgram/kg and was maximal at 10 micrograms/kg. Treatment of sensitized mice with salbutamol increased the ex vivo production of IL-4, IL-5, IL-6 and IL-10 from concanavalin A-activated splenocytes whereas no modification of IFN-gamma synthesis was noticed as compared to nontreated sensitized control mice. These results demonstrate that beta 2-adrenoceptor agonist stimulation results in an increase in IgE production both in vitro and in vivo in the mice. At least in vivo, they also suggest that the effect of this drug could be explained by an increase of the production of Th2-type lymphokines.
Assuntos
Albuterol/farmacologia , Linfócitos B/efeitos dos fármacos , Imunoglobulina E/biossíntese , Animais , Antígenos de Superfície/biossíntese , Linfócitos B/imunologia , Células Cultivadas , Concanavalina A/imunologia , Citocinas/biossíntese , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Baço/citologiaRESUMO
Interleukin 4 (IL-4)-induced IgE production by normal peripheral blood mononuclear cells (PBMC) and E- cells (PBMC partially depleted of T cells) was significantly enhanced by leukotriene B4(LTB4) in a dose-dependent manner, whereas LTB4 by itself was not effective for IgE production. The potentiating effect of LTB4 was strictly dependent on IL-4. When PBMC or E- cells were primed with IL-4 (300 U/ml) for 48 hr, then recultured with LTB4 alone (10(-10) to 10(-8) M), increased IgE production was observed. Maximum enhancement of IgE production after IL-4 priming was achieved using a combination of IL-4 and LTB4, acting additively. Moreover, the potentiating effect of LTB4 on IL-4-induced IgE production was completely dependent on the presence of a monocyte/macrophage population. This effect of LTB4 was completely abolished by depletion of monocytes and recovered by reconstitution with autologous monocytes. From the study of IL-4 receptor (IL-4R) expression determined by flow cytometry, IL-4 was found to upregulate the biotinylated-IL-4 (B-IL-4)/streptavidin binding to both T- and B-cell populations. A further increase of B-IL-4 binding was obtained when the cells were incubated with IL-4 and LTB4. Finally, LTB4 can induce soluble CD23 (sCD23) release by E- cells tested either alone or in the presence of IL-4. Taken together, these data suggest that LTB4 can influence the IL-4-induced IgE production through, at least, a bimodal action, i.e., increase of IL-4R positive cells and release of sCD23. These findings may be a specific feature of LTB4 which provides a crucial role in IL-4-linked allergic inflammatory process.
Assuntos
Imunoglobulina E/biossíntese , Interleucina-4/farmacologia , Leucotrieno B4/farmacologia , Linfócitos/metabolismo , Humanos , Macrófagos/imunologia , Monócitos/imunologia , Receptores de IgE/química , Receptores de IgE/metabolismo , Receptores de Interleucina-4 , Receptores Mitogênicos/metabolismo , SolubilidadeRESUMO
Human bone-marrow or cord-blood progenitors (i.e., CD34+ cells) are easily purified by immunological methods and can be cultured on normal human-bone-marrow stromal cells for limited periods of time. Under these culture conditions, the number of progenitors declines in a few weeks and these cells disappear completely in less than 8 weeks. This fact suggests that this culture system is deprived of growth factor(s) able to support the self-renewal of stem cells. We have developed the culture of immunomagnetically purified human-bone-marrow- or cord-blood-derived CD34+ cells on a supportive mouse lipoblastic stromal cell line, MS-5. The long-term survival of clonogenic cells was analyzed in these cultures and compared with the results obtained by culture on human-bone-marrow stromal cells. The results demonstrated that only coculture of CD34+ cells on MS-5 layers allows the survival of clonogenic progenitors for at least 12 weeks. Cytospin smears were regularly performed and cell morphology was examined after classical staining methods (i.e., M.G.G. and toluidine blue staining). Histologic analysis demonstrated the growth of mast-cell-like metachromatic cells after the second week of incubation on MS-5 layer. The highest percentage of these cells was observed after 8 weeks, and averaged about 30 percent for cord-blood cells and 70 percent for bone-marrow cells. To further confirm the nature of the metachromatic cells obtained under this culture condition, immunohistochemical staining of tryptase was performed on the same samples. The results demonstrated similar percentages of tryptase+ cells and of metachromatic elements. Measurement of cellular histamine demonstrated that culture of CD34+ cells on MS-5 monolayers induced the formation and increase of this mediator. To determine whether the contact between MS-5 layers and CD34+ cells was an absolute requirement for the development of mast cells, CD34+ cells were cultured in the presence of MS-5 conditioned medium. This condition allowed the development of similar percentage of mast cells when compared with the coculture experiments, indicating that a soluble factor was involved in mast cell differentiation. Whatever the soluble factor(s) responsible for this mast cell growth activity, our culture system allows us to obtain significant amounts of highly enriched normal human mast cell populations useful for further studies on the reactivity of this cell subset.
Assuntos
Células da Medula Óssea , Sangue Fetal/citologia , Mastócitos/citologia , Células-Tronco/citologia , Células Estromais/metabolismo , Animais , Antígenos CD/análise , Antígenos CD34 , Moléculas de Adesão Celular , Diferenciação Celular , Linhagem Celular , Meios de Cultivo Condicionados , Fatores de Crescimento de Células Hematopoéticas , Histamina/metabolismo , Humanos , Leucócitos Mononucleares , Camundongos , Fator de Células-Tronco , Células-Tronco/fisiologiaRESUMO
The effect of the immunosuppressive compound, cyclosporin A, and the corticosteroid, betamethasone, was investigated on eosinophil accumulation in guinea-pig lung tissue induced by antigen, platelet-activating factor (PAF) and leukotriene B4 (LTB4). The accumulation of eosinophils in the peribronchial area was evaluated on histological preparations. The lung sections were stained with Luna's reagent specific for eosinophil granule content. Oral treatment of the guinea-pigs with cyclosporin, 10 mg.kg-1 three times a day for two days, and 10 mg.kg-1 1 h before antigen challenge, significantly reduced the accumulation of eosinophils observed at 4 and 24 h, in the peribronchial area of sensitized guinea-pig lung. Betamethasone (3 mg.kg-1), administered orally 24 h and 1 h before antigen challenge elicited a moderate but significant reduction of antigen-induced eosinophil accumulation. Pretreatment of the guinea-pigs with cyclosporin or betamethasone elicited a marked inhibition of the accumulation of eosinophils in the peribronchial area induced by aerosolized PAF (100 micrograms.ml-1) or LTB4 (5 micrograms.ml-1). Since cyclosporin and betamethasone significantly inhibit the antigen-induced eosinophil accumulation, these results suggest that antigen-induced lung eosinophilia is dependent of T-lymphocytes. However, cyclosporin and betamethasone may also reduce the chemotactic activity of PAF and LTB4 on guinea-pig eosinophils.
Assuntos
Antígenos/imunologia , Ciclosporina/farmacologia , Eosinófilos/patologia , Leucotrieno B4/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Fator de Ativação de Plaquetas/farmacologia , Animais , Betametasona/farmacologia , Ciclosporina/administração & dosagem , Cobaias , Pulmão/patologia , Masculino , Eosinofilia Pulmonar/patologiaAssuntos
Agonistas Adrenérgicos beta/farmacologia , Albuterol/farmacologia , Imunoglobulina E/biossíntese , Leucócitos Mononucleares/imunologia , Animais , Butoxamina/farmacologia , Células Cultivadas , Citocinas/imunologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Propranolol/farmacologia , Baço/citologiaRESUMO
The beta 2-adrenoceptor agonists salbutamol and fenoterol were tested for their regulatory effects on human monocyte phenotype and functions, either alone or in combination with interleukin-4 (IL-4). These drugs enhanced in a dose-dependent manner the IL-4-induced membrane and mRNA expression of the low-affinity receptor for immunoglobulin E (IgE) (CD23), as well as the release of its soluble form, sCD23. Salbutamol and fenoterol alone elicited expression of the monomorphic beta 2-chain (CD18) of the leukocyte functional antigen (LFA1) family. This effect appeared to be restricted to CD11b (CR3) and CD11c (gp 150-95), because CD11a (LFA-1 alpha chain) was not modified. beta 2-Adrenoceptor stimulation was also found to potentiate the effect of IL-4 on CD11b, CD11c, and CD18 expression. In contrast, these agents alone did not alter the level of major histocompatibility complex class II and CD14 antigens or modify their respective up- and down-regulation by IL-4. Ligation of CD23 on IL-4-preincubated (CD23+) monocytes with IgE/anti-IgE immune complexes induced the release of free radicals nitric oxide and of the proinflammatory mediators IL-6 and thromboxane B2 (TxB2). Addition of salbutamol, inactive alone, potentiated the generation of superoxide anion and of nitric oxide generation, as well as the production of IL-6 and TxB2 triggered by CD23 ligation. These results indicate that beta 2-adrenoceptor stimulation potentiates in vitro the IL-4-induced phenotypical and functional changes on monocytes and suggest that such an interaction could occur in IgE-dependent immune reactions.
Assuntos
Imunoglobulina E/farmacologia , Interleucina-4/farmacologia , Monócitos/citologia , Monócitos/fisiologia , Receptores Adrenérgicos beta/fisiologia , Albuterol/farmacologia , Antígenos CD/análise , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/análise , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Northern Blotting , Células Cultivadas , Fenoterol/farmacologia , Imunofluorescência , Antígenos de Histocompatibilidade Classe II/análise , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Interleucina-6/metabolismo , Receptores de Lipopolissacarídeos , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/metabolismo , Antígeno-1 Associado à Função Linfocitária/fisiologia , Monócitos/química , Fenótipo , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores de IgE/genética , Receptores de IgE/metabolismo , Receptores de IgE/fisiologia , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Tromboxano B2/metabolismoRESUMO
The involvement of platelet activating factor (PAF) in antigen-induced bronchial hyperresponsiveness was investigated by the use of the PAF antagonists BN 52021 and BN 50730, in a guinea-pig model where sensitization and challenge were performed by aerosol. Male Hartley guinea-pigs were sensitized by two aerosol exposures at 48 hr intervals to a 0.9% NaCl solution (saline) containing 2 mg/ml ovalbumin for 30 min. Fifteen to 20 days later, guinea-pigs were challenged by exposure to five successive aerosols of increasing concentrations of ovalbumin (OA) or respectively, 10 microg/ml, 100 microg/ml, 1 mg/ml, 5 mg/ml and 10 mg/ml for 15 min each, or saline alone. Three to four hr and 18-24 hr after the aerosol challenge the guinea-pigs were prepared for recording of bronchopulmonary response and aerosol administrations were then generated with an ultrasonic nebulizer. The bronchopulmonary responses induced by successive 1-min aerosol bursts of acetylcholine (ACh) was assessed. As compared with saline-challenged guinea-pigs, an enhanced bronchopulmonary response to aerosol administration of cumulative doses of ACh was observed, 3-4 hr and 18-24 hr post-ovalbumin challenge. When the sensitized guinea-pigs were pretreated 1 hr before ovalbumin exposure with BN 52021 or BN 50730 (25 mg/kg, per os), a significant inhibition of the increase in the bronchopulmonary response to ACh was observed, both at 3-4 hr and 18-24 hr. Furthermore, when guinea-pigs were treated 3-4 hr after the ovalbumin exposure with BN 52021 or BN 50730, a significant inhibition of the hyperresponsiveness to ACh was recorded at 18-24 hr. A marked accumulation of eosinophils in the peribronchial regions was observed on histological preparations of lung specimens collected 4 hr or 24 hr after ovalbumin exposure. Pretreatment of the guinea-pigs by BN 50730 or BN 52021 did not modify the eosinophil accumulation in the peribronchial area. No significant difference in the number of eosinophils collected in the bronchoalveolar lavage fluid is observed, 24 hr post-ovalbumin challenge, under the pretreatment with BN 52021 or BN 50730. Pretreatment of guinea-pigs by BN 50730 or BN 52021 significantly reduced the PAF-induced (100 microg/ml) increase in eosinophil number in the peribronchial area. By contrast, they did not inhibit the eosinophilia induced by aerosol administration of LTB4 (5 microg/ml). These results suggest that the bronchial hyperresponsiveness observed in this study is associated with eosinophil accumulation in the lung. The potent inhibition of the bronchial hyperresponsiveness by the two unrelated antagonists of PAF suggests that the lipid mediator is involved in its triggering and duration, but not in the eosinophil infiltration.