RESUMO
Binding sites for the nuclear factor (NF)-kappaB transcription factor have been identified within control regions of many genes involved in inflammatory and immune responses. Such kappaB sites are often found adjacent to those of interferon (IFN)-gamma-inducible transcription factors, suggesting a requirement for multiple signaling pathways for gene regulation. Using fibroblasts from RelA (p65)-deficient mice generated by gene targeting, we have investigated the role of this subunit of NF-kappaB in gene activation by microbial lipopolysaccharide, tumor necrosis factor alpha, and in possible synergism with the IFN-gamma-signaling pathway. Our results indicate not only that RelA is required for activation of key genes involved in adaptive (acquired) immune responses, including major histocompatibility complex class I, CD40, and the Fas death receptor, but also that both NF-kappaB-inducing signals and IFN-gamma are necessary for maximal activation. In contrast, neutrophil-specific chemokine genes KC and MIP-2, which can function as nonspecific mediators in innate immune responses, were strongly induced by RelA in the absence of IFN-gamma. Our results show that RelA plays a critical role in activation of immune system genes in response to nonspecific stimuli and demonstrate a novel proapoptotic function for this protein in Fas-induced cell death.
Assuntos
Apoptose , Regulação da Expressão Gênica , Genes MHC da Classe II , Ligases/fisiologia , NF-kappa B/fisiologia , Receptor fas/fisiologia , Animais , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Ativação Transcricional , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Epidermal keratinocytes (EK) are exposed to multiple inflammatory stimuli and paracrine factors secreted by various dermal cells (lymphocytes, mast cells, macrophages, fibroblasts) during wounding, cutaneous allergy, and infections. We have previously demonstrated that after stimulation with interleukin 4 or interferon-gamma, human EK express the low-affinity receptor for IgE (Fc epsilon RII/CD23) on their surface. In the present study, we showed that the ligation of CD23 by IgE/anti-IgE immune complexes or specific monoclonal antibody induces a dose-dependent release of interleukin 6 and tumor necrosis factor-alpha from EK. CD23-ligation activates the nitric oxide-dependent pathway, as demonstrated by the high levels of nitrites released in cell supernatants, and the accumulation of intracellular cyclic nucleotides in EK. These second messengers are required for IgE-dependent stimulation of cytokine production by these cells, inasmuch as this is completely abolished by the use of cAMP or nitric oxide synthase antagonists. Human epithelial keratinocytes may thus participate in IgE-mediated immune responses, through their ability to express functional CD23 antigen.
Assuntos
AMP Cíclico/metabolismo , Imunoglobulina E/farmacologia , Queratinócitos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Receptores de IgE/metabolismo , Anticorpos Monoclonais/farmacologia , Arginina/metabolismo , Divisão Celular/efeitos dos fármacos , Separação Celular , Células Cultivadas , GMP Cíclico/metabolismo , Humanos , Imunoglobulina E/imunologia , Imuno-Histoquímica , Hibridização In Situ , Recém-Nascido , Interleucina-6/metabolismo , Masculino , Nitratos/metabolismo , RNA Mensageiro/isolamento & purificação , Receptores de IgE/genética , Receptores de IgE/imunologia , Transdução de Sinais/efeitos dos fármacos , Pele/citologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Transduction through Fc epsilon R2/CD23 was analyzed in normal human monocytes using immunoglobulin E (IgE)-anti-IgE immune complexes (IgE ICs) and monoclonal antibodies (mAbs) to CD23. Anti-CD23 mAb and IgE IC triggered a time-dependent increase in cGMP and cAMP in interleukin-4-preincubated (CD23+) but not in unstimulated (CD23-) monocytes. Maximal cGMP and cAMP accumulations were observed 10 and 20 min, respectively, after the onset of CD23 ligation. The increase in cGMP was inhibited with N omega-monomethyl-L-arginine (L-NMMA), which also partially affected cAMP accumulation. Addition of an anti-CD23 mAb Fab fragment inhibited the IgE IC- and the anti-CD23 mAb-induced cGMP and cAMP accumulation, confirming the engagement of CD23. In addition, IgE IC and anti-CD23 mAb induced, at least in some donors, a production of nitrite that was inhibited in the presence of L-NMMA. Taken together, these findings suggest a possible involvement of the nitric oxide synthase pathway in IgE IC-mediated activation of CD23+ monocytes.
Assuntos
Arginina/fisiologia , Guanilato Ciclase/metabolismo , Monócitos/enzimologia , Receptores de IgE/fisiologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Complexo Antígeno-Anticorpo/farmacologia , Arginina/análogos & derivados , Arginina/farmacologia , Células Cultivadas , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Ativação Enzimática , Guanilato Ciclase/fisiologia , Humanos , Imunoglobulina E/farmacologia , Interleucina-4/farmacologia , Monócitos/citologia , Monócitos/fisiologia , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/metabolismo , Receptores de IgE/imunologia , ômega-N-MetilargininaRESUMO
CD23 is a surface antigen expressed by a variety of human hematopoietic cells and shown to display multiple biological functions. In present work, we assayed CD23 expression by human bone marrow (BM) or by stromal cells derived from this tissue. While freshly isolated BM-cells showed low CD23 expression, a subset of long term BM-culture (LTBMC)-derived stromal cells expressed CD23 mRNA at high levels in their steady state and secreted soluble CD23 in their culture supernatants. To assay the role of CD23 in LTBMC, these cultures were initiated in the presence of neutralizing anti-CD23 mAb. A dramatic decrease in total numbers of hematopoietic cells and CFU-GM recovery was observed in these cultures as compared to controls. These data suggest a role of CD23 expression in stroma cell functions and further confirm the ability of this antigen to regulate human hematopoietic cell development.
Assuntos
Medula Óssea/imunologia , Receptores de IgE/biossíntese , Anticorpos Monoclonais , Células da Medula Óssea , Expressão Gênica , Hematopoese/imunologia , Humanos , Hibridização In Situ , Técnicas In Vitro , RNA Mensageiro/genética , Receptores de IgE/antagonistas & inibidores , Receptores de IgE/genéticaRESUMO
In a case of indolent stage A chronic lymphocytic leukemia (C.L.L.), treated for ten years only by theophylline for bronchial asthma, we observed spontaneous apoptosis of B lymphocytes (10%). As suggested by these case report, we described new properties of methylxanthine derivatives. In vitro, theophylline increased spontaneous apoptosis after 72 hours in culture of 6 patients by a mean percentage of 80-90% in B-C.L.L. blood lymphocytes (control 20%). Dose-dependent apoptosis involves cyclic nucleotides (AMPc). Using identical theophylline doses, we did not observe apoptosis of normal peripheral blood B lymphocytes. According to French ethical rules, we treated 8 patients with the same doses of theophylline than for bronchial asthma without responses. On the other hand, in 12 aggressive forms of C.L.L., resistant or in relapsed after alkylating agents, methylxanthine derivatives appeared a powerful adjuvant of chlorambucil treatment. We observed 11 responses with less dose of alkylating agents than in previous treatment: decrease in the concentration of blood lymphocytes (11 patients) and clinical remissions (8 patients). Mechanism of action and future of this new drugs combination in the treatment of C.L.L. are discussed.
Assuntos
Antineoplásicos Alquilantes/administração & dosagem , Apoptose/efeitos dos fármacos , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Broncodilatadores/administração & dosagem , Broncodilatadores/farmacologia , Clorambucila/administração & dosagem , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Pentoxifilina/farmacologia , Teofilina/administração & dosagem , Teofilina/farmacologia , Idoso , Asma/tratamento farmacológico , Quimioterapia Combinada , Feminino , Humanos , Técnicas In Vitro , Masculino , Pentoxifilina/administração & dosagem , Indução de Remissão , Fatores de Tempo , Células Tumorais Cultivadas , Vasodilatadores/administração & dosagem , Vasodilatadores/farmacologiaRESUMO
During intrathymic T cell development, elimination of autoreactive T cell clones by programmed cell death (PCD or apoptosis) is an essential mechanism for self tolerance. The precise intracellular second messengers that lead to this process remain to be determined. In the present work, we show that treatment of freshly isolated thymocytes with an antagonist of the cAMP pathway, the Rp-cAMP, significantly decreases spontaneous death by apoptosis of human thymocytes in vitro. Addition of Rp-cAMP also rescues thymocytes from activation-induced apoptosis following the ligation of surface CD3/T cell receptor complex or CD2 antigens. A cAMP analog, the dibutyryl(Dibut)-cAMP increases PCD of human thymocytes in a dose-dependent manner. Growth and rescue from PCD of thymocytes in the presence of interleukin (IL)-2 or IL-4 are also enhanced by Rp-cAMP and inhibited by Dibut-cAMP. Finally, we detect substantial levels of intracellular cAMP in freshly isolated thymocytes. This study reveals the involvement of cAMP as a second messenger during the apoptosis of normal human thymocytes.
Assuntos
Apoptose , Antígenos CD2/metabolismo , Complexo CD3/metabolismo , AMP Cíclico/metabolismo , Linfócitos T/patologia , Divisão Celular , Células Cultivadas , Humanos , Linfócitos T/metabolismoRESUMO
The low-affinity receptor for IgE (CD23) is cleaved into biologically active soluble fragments (sCD23), some of which have been reported to exhibit pleiotropic activities. However, it is not known whether the sCD23 fragments contribute to the induction and/or regulation of pro-inflammatory cytokine production. In this study, this possibility was tested using interleukin (IL)-1-stimulated human whole blood as an ex vivo model of cytokine cascade production. We show that human recombinant 25-kDa sCD23 significantly enhanced the production of IL-6 in whole blood stimulated by IL-1, but had only little or no effect in the absence of IL-1. The potentiating effect of sCD23 was concentration dependent within the range of plasma levels occurring during various inflammatory processes in man. These results prompted us to study whether sCD23 and IL-1 together also enhance the production of regulating factors exhibiting anti-cytokine activities. Our data indicate that sCD23 augments the release of IL-1 receptor antagonist induced by IL-1. Finally, examining the effect of sCD23 on human peripheral monocytes stimulated by IL-1, we confirmed the capacity of sCD23 to potentiate cytokine production. We suggest that sCD23 can modulate monocyte functions, thereby contributing to the amplification and regulation of immune and inflammatory processes.
Assuntos
Interleucina-1/fisiologia , Interleucina-6/biossíntese , Monócitos/imunologia , Receptores de IgE/fisiologia , Sialoglicoproteínas/biossíntese , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas In Vitro , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Receptores de IgE/química , Receptores de Interleucina-1/antagonistas & inibidores , Sialoglicoproteínas/metabolismoRESUMO
Ligation of the low affinity IgE receptor by specific monoclonal antibodies or multivalent IgE complexes result in the transduction of signals which differ according to the CD23 isotype expressed by the various cell types. In B lymphocytes, it elicits the early activation of phospholipase C through a mechanism involving a G-protein insensitive to Pertussis toxin, followed by a late phase of cAMP accumulation. In monocytes, which express the CD23b isoform, ligation of CD23 was also found to induce a delayed accumulation of cAMP, that was largely dependent on a prior cGMP increase through a mechanism involving the activation of a NO synthase. This pathway, which appears to be exacerbated in allergic diseases, seems to play an important role in the differentiation of cells of the monocytic lineage, their capacity to release proinflammatory mediators and their cytotoxic functions.
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Receptores de IgE/fisiologia , Aminoácido Oxirredutases/metabolismo , Linfócitos B/fisiologia , GMP Cíclico/biossíntese , Técnicas In Vitro , Monócitos/metabolismo , Monócitos/fisiologia , Óxido Nítrico Sintase , Receptores de IgE/química , Receptores de IgE/metabolismo , Transdução de SinaisRESUMO
The Fas death receptor plays a key role in the killing of target cells by NK cells and CTLs and in activation-induced cell death of mature T lymphocytes. These cytotoxic pathways are dependent on induction of Fas expression by cytokines such as TNF-alpha and IFN-gamma or by signals generated after TCR engagement. Although much of our knowledge of the Fas death pathway has been generated from murine studies, little is known about regulatory mechanisms important for murine Fas expression. To this end, we have molecularly cloned a region of the murine Fas promoter that is responsible for mediating TNF-alpha and PMA/PHA-induced expression. We demonstrate here that induction of Fas expression by both stimuli is critically dependent on two sites that associate with RelA-containing NF-kappaB complexes. To determine whether RelA and/or other NF-kappaB subunits are also important for regulating Fas expression in primary T cells, we used CD4 T cells from RelA(-/-), c-Rel(-/-), and p50(-/-) mice. Although proliferative responses were significantly impaired, expression of Fas and activation-induced cell death was unaffected in T cells obtained from these different mice. Importantly, we show that unlike fibroblasts, which consist primarily of RelA-containing NF-kappaB complexes, T cells have high levels of both RelA and c-Rel complexes, suggesting that Fas expression in T cells may be dependent on redundant functions of these NF-kappaB subunits.
Assuntos
Apoptose/imunologia , Ativação Linfocitária , NF-kappa B/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Receptor fas/biossíntese , Células 3T3 , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Sequência de Bases , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Clonagem Molecular , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Proteína Ligante Fas , Fibroblastos/metabolismo , Humanos , Células Jurkat , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , NF-kappa B/deficiência , NF-kappa B/genética , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B , Fito-Hemaglutininas/farmacologia , Regiões Promotoras Genéticas/imunologia , Proteínas Proto-Oncogênicas c-rel/deficiência , Proteínas Proto-Oncogênicas c-rel/genética , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição RelA , Receptor fas/genética , Receptor fas/metabolismoRESUMO
We report a case of indolent B-chronic lymphocytic leukaemia (B-CLL) in a stage A patient, treated for 10 years only by theophylline for bronchial asthma. As suggested by the spontaneous apoptosis in the patient's blood (10%), theophylline at 50 micrograms/ml increased spontaneous apoptosis after 72 h in culture by a mean percentage of 90% (range 79-98%) in six B-CLL cases studied in vitro. This effect was partially reversed with Rp-cAMP, a cAMP antagonist, which implies a potent role for this second messenger. We describe a new property of theophylline, which might be an alternative treatment in B-CLL.
Assuntos
Apoptose/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Teofilina/farmacologia , Idoso , Linfócitos B/patologia , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Linfopenia , Tionucleotídeos/farmacologiaRESUMO
The early events triggered in interleukin-4 (IL-4)-stimulated U937 cells by ligation of CD23/Fc epsilon RII with specific monoclonal antibodies (mAb) were analysed, as a model of the action of this molecule on the differentiation of promonocytic cells. As well as IL-4-activated human monocytes, addition of anti-CD23 mAb to IL-4-treated U937 cells triggered cAMP accumulation but did not evoke significant polyphosphoinositide hydrolysis. However, by a microspectrofluorometric technique allowing single cell analysis, anti-CD23 mAb was found to elicit calcium mobilization in these cells. In addition, the treatment induced phenotypic alterations in these cells, as evidenced by the acquisition of the monocyte marker CD14 and the increase of the alpha-chain (CD11a) and of the common beta-chain (CD18) of the leucocyte function-associated antigen 1 (LFA-1) family antigens. Although weaker than in monocytes, CD23 ligation evoked a small secretion of the pro-inflammatory mediators IL-6 and thromboxane B2. These data suggest that a significant maturation of promonocytic cells towards a more mature monocytic phenotype can be achieved through successive exposure to IL-4 and CD23 ligation.
Assuntos
Monócitos/imunologia , Receptores de IgE/imunologia , Anticorpos Monoclonais/imunologia , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Antígenos CD11 , Antígenos CD18 , Cálcio/metabolismo , Divisão Celular/imunologia , AMP Cíclico/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/biossíntese , Interleucina-4/imunologia , Interleucina-6/biossíntese , Leucemia Monocítica Aguda/imunologia , Receptores de Lipopolissacarídeos , Tromboxano B2/biossíntese , Células Tumorais CultivadasRESUMO
Serum IgE concentrations and the expression of the low-affinity receptor for IgE (Fc epsilon RII/CD23) are increased in cutaneous leishmaniasis or after immune challenge with Leishmania antigens. In vitro, the ligation of CD23 by IgE-anti-IgE immune complexes (IgE-IC) or by anti-CD23 monoclonal antibody (mAb) induces nitric oxide (NO) synthase and the generation of various cytokines by human monocytes/macrophages. The present study shows that IgE-IC, via CD23 binding, induce intracellular killing of Leishmania major in human monocyte-derived macrophages through the induction of the L-arginine:NO pathway. This was demonstrated by increased generation of nitrite (NO2-), the stable oxidation product of NO, and by the ability of NG-monomethyl-L-arginine to block both NO generation and parasite killing. A similar NO-dependent effect was observed with interferon gamma-treated cells. Tumor necrosis factor alpha is involved in this process, since both the induction of NO synthase and the killing of parasites caused by anti-CD23 mAb were inhibited by an anti-tumor necrosis factor alpha mAb. Treatment of noninfected CD23+ macrophages with IgE-IC provided protection against subsequent in vitro infection of these cells by Leishmania major promastigotes. Thus, IgE-IC promote killing of L. major by inducing NO synthase in human macrophages.
Assuntos
Aminoácido Oxirredutases/biossíntese , Citocinas/farmacologia , Leishmania major/imunologia , Macrófagos/imunologia , Óxido Nítrico/biossíntese , Receptores de IgE/fisiologia , Aminoácido Oxirredutases/antagonistas & inibidores , Aminoácido Oxirredutases/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Antígenos CD/análise , Antígenos CD/fisiologia , Arginina/análogos & derivados , Arginina/farmacologia , Sequência de Bases , Células Cultivadas , Primers do DNA , Expressão Gênica , Humanos , Interferon gama/farmacologia , Interleucina-4/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Dados de Sequência Molecular , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico Sintase , Fagocitose , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Proteínas Recombinantes/farmacologia , Superóxidos/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , ômega-N-MetilargininaRESUMO
BACKGROUND: IgE/anti-IgE immune complexes (IgE-IC) induce the release of multiple mediators from monocytes/macrophages and the monocytic cell line U937 following the ligation of the low-affinity Fc epsilon receptors (Fc epsilon RII/CD23). These effects are mediated through an accumulation of cAMP and the generation of L-arginine-dependent nitric oxide (NO). Since high IgE levels predict more rapid progression to acquired immunodeficiency syndrome, we attempted to define the effects of IgE-IC on human immunodeficiency virus (HIV) production in monocytes. MATERIALS AND METHODS: Two variants of HIV-1 chronically infected monocytic U1 cells were stimulated with IgE-IC and virus replication was quantified. NO and cAMP involvement was tested through the use of agonistic and antagonistic chemicals of these two pathways. RESULTS: IgE-IC induced p24 production by U1 cells with low-level constitutive expression of HIV-1 mRNAs and extracellular HIV capsid protein p24 levels (U1low), upon their pretreatment with interleukin 4 (IL-4) or IL-13. This effect was due to the crosslinking of CD23, as it was reversed by blocking the IgE binding site on CD23. The IgE-IC effect could also be mimicked by crosslinking of CD23 by a specific monoclonal antibody. p24 induction by IgE-IC was then shown to be due to CD23-mediated stimulation of cAMP, NO, and tumor necrosis factor alpha (TNF alpha) generation. In another variant of U1 cells with > 1 log higher constitutive production of p24 levels (U1high), IgE-IC addition dramatically decreased all cell functions tested and accelerated cell death. This phenomenon was reversed by blocking the nitric oxide generation. CONCLUSIONS: These data point out a regulatory role of IgE-IC on HIV-1 production in monocytic cells, through CD23-mediated stimulation of cAMP and NO pathways. IgE-IC can also stimulate increased cell death in high HIV producing cells through the NO pathway.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Morte Celular/genética , HIV-1/genética , Imunoglobulina E/imunologia , Monócitos/metabolismo , Complexo Antígeno-Anticorpo/farmacologia , AMP Cíclico/farmacologia , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Regulação da Expressão Gênica/genética , HIV-1/metabolismo , Humanos , Imunoglobulina E/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacologia , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptores de IgE/metabolismo , Transdução de Sinais , Replicação Viral/genéticaRESUMO
Early thymocytes undergo extensive proliferation after their entry into the thymus, but cellular interactions and cytokines regulating this intrathymic step remain to be determined. We analyzed the effects of various T-cell growth factors and cellular interactions on in vitro proliferation of early CD2+CD3/TCR-CD4-CD8- (triple negative [TN]) human thymocytes. Freshly isolated TN cells were then assayed for their growth capacity after incubation with CD2I+III-monoclonal antibody (MoAb), recombinant human interleukin-2 (IL-2), IL-7, and/or IL-4. These cells displayed significant proliferative responses with IL-4, IL-7, or CD2-MoAb+IL-2. The addition of recombinant transforming growth factor beta (TGF beta) or autologous irradiated CD3+CD8+CD4- cells to TN cell cultures dramatically decreased their growth responses to IL-2 and IL-7, whereas IL-4-induced proliferation was less sensitive to growth inhibition. We thus asked whether the CD8+ cell-derived inhibitory effect was due to TGF beta. The addition of neutralizing anti-TGF beta MoAb completely abolished CD8+ cell-derived inhibition of TN cell growth. Analysis of CD8+ cell-derived supernatants indicated that these cells had low TGF beta 1 production capacity, whereas TN cells secrete significantly high levels of TGF beta 1. Cell fixation studies showed that TN cells were the source of the TGF beta. TGF beta 1 released from TN cells was in the latent form that became the active inhibitory form through interaction of TN cells with CD8+ cells. Together, these data suggest a role for TGF beta 1 as an externally controlled, autocrine inhibitory factor for human early thymocytes, with a regulatory role in thymic T-cell output.
Assuntos
Antígenos CD4/biossíntese , Antígenos CD8/biossíntese , Linfócitos T/citologia , Linfócitos T/metabolismo , Timo/citologia , Fator de Crescimento Transformador beta/biossíntese , Comunicação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Pré-Escolar , Humanos , Imunofenotipagem , Lactente , Interleucinas/farmacologia , Subpopulações de Linfócitos T , Fator de Crescimento Transformador beta/farmacologiaRESUMO
The regulation of the low affinity receptor for IgE (Fc epsilon RII/CD23) expression and its role were investigated in U937 cell line and in leukemic cells from a patient (Amb) with acute myeloblastic leukemia. Both cell populations were CD23- but could acquire CD23 expression following treatment with IL-4. CD23+ cells, however, remained blastic and did not show any significant phenotypical and functional modifications. Following ligation of the CD23 on U937 and Amb cells by anti-CD23 mAb, these leukemic cells differentiated into mature monocyte/macrophage-like cells. CD23 ligation promoted the expression of the monocyte marker, CD14, increased the expression of the common beta chain of the LFA-1 family (CD18), and down-regulated the expression of the promonocytic marker CD33. Morphological and phenotypical changes were associated with functional modifications as CD23 ligation allowed the acquisition of the oxidative metabolism in leukemic cells as revealed by luminol-dependent chemiluminescence. As in mature monocytes, CD23 ligation induced an accumulation of intracellular cAMP in leukemic cells. These data indicate that ligation of CD23 may induce the maturation of myelomonocytic cells into monocytic-like cells.
Assuntos
Leucemia Mielomonocítica Aguda/patologia , Receptores de IgE/fisiologia , AMP Cíclico/biossíntese , Humanos , Interleucina-4/farmacologia , Proteínas Recombinantes/farmacologia , Explosão Respiratória , Células Tumorais CultivadasRESUMO
The effects of interleukin 4 (IL-4) on human leukemic precursor B-cell lines were investigated. Recombinant IL-4 (rIL-4) was added to three acute lymphoblastic leukemia-derived pre B-cell lines: Reh, Km3 and Nalm-6. Our results show that rIL-4 significantly decreases continuous proliferation of Reh and Km3 cells while Nalm-6 cells have a limited response in this respect. This rIL-4 effect is dose-dependent and can be neutralized by anti-IL-4 monoclonal antibody (mAb). Furthermore, rIL-4 down-regulated IL-3-induced proliferation of Reh cells. Phenotypic analysis of rIL-4-treated cells points to significant induction of surface marker maturation of leukemic cells by this cytokine. Together, these in vitro data suggest that IL-4: 1) inhibits the proliferation and 2) promotes the differentiation of certain human leukemic B-cell precursors.
Assuntos
Linfócitos B/patologia , Divisão Celular , Interleucina-4/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Antígenos de Diferenciação de Linfócitos B/análise , Células-Tronco Hematopoéticas/patologia , Humanos , Interleucina-2/farmacologia , Interleucina-3/farmacologia , Proteínas Recombinantes/farmacologia , Células Tumorais CultivadasRESUMO
Recently, authors have addressed the ability of human basophils to produce IL-4. We report here the detection of significant serum IL-4 levels in a case of acute transformation of chronic myelogenous leukemia with a predominant basophilic cell population. Leukemic basophils were isolated from patients' PBMC and assayed for their IL-4-mRNA expression and their ability to secrete this cytokine in vitro. Leukemic basophilic cells (> 90% toluidine blue positive) but not other PBMC expressed IL-4-mRNA, contained IL-4 protein, and secreted this cytokine. These cells had a spontaneous IL-4 secretion ability, without a need for an exogenous activator. Meanwhile, IL-4 release was significantly increased following leukemic cell activation through Fc epsilon RI-ligation or by Ca2+ ionophore. IL-4 and its mRNA were also detected in leukemic basophils from three other chronic myelogenous leukemia patients with moderate basophilia (13, 14, and 23% basophils in PBMC). To confirm these data in normal human cells, we have developed a method to obtain large numbers of purified basophils from human bone marrow cell cultures. In contrast to leukemic basophils, normal cells required in vitro activation through Fc epsilon RI ligation or by Ca2+ ionophore to express and secrete IL-4. Leukemic and normal basophils secreted histamine following in vitro activation, but were negative for tryptase. These data thus demonstrate the in vivo and in vitro ability of human basophils to produce IL-4.
Assuntos
Basófilos/imunologia , Interleucina-4/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Basófilos/citologia , Células da Medula Óssea , Separação Celular , Expressão Gênica , Liberação de Histamina , Humanos , Hibridização In Situ , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , RNA Mensageiro/genéticaRESUMO
Elevated IgE levels are commonly observed during the inflammatory responses in allergy and a variety of infections. This Ig activates the release of multiple mediators from monocytes/macrophages. In the present work, we attempted to clarify the IgE-dependent events involved in the activation of monocyte functions. IgE-anti-IgE immune complexes induce the production of tumor necrosis factor-alpha, oxygen radicals, IL-6 and thromboxane B2 from normal human purified monocytes. Expression and cross-linkage of Fc epsilon RII/CD23 were essential for these IgE-mediated effects. Cytokine production following CD23 ligation depended on nitric oxide transduction pathway, as it was inhibited by NG-monomethyl-L-arginine, a competitive inhibitor of the conversion of L-arginine to L-citroline by nitric oxide synthase. Furthermore, addition of the nitric oxide chemical donator, Sin-1, enhanced IgE-induced monokine release. CD23-ligation also induced the production of nitrites by these cells. This work linked CD23 to the L-arginine-dependent transduction pathway and shows their involvement in IgE-mediated stimulation of human monocytes.
Assuntos
Arginina/imunologia , Imunoglobulina E/imunologia , Monócitos/imunologia , Receptores de IgE/imunologia , Transdução de Sinais/imunologia , Arginina/análogos & derivados , Arginina/farmacologia , Células Cultivadas , Citocinas/biossíntese , Humanos , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Monócitos/efeitos dos fármacos , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/fisiologia , Superóxidos/metabolismo , Tromboxano B2/biossíntese , ômega-N-MetilargininaRESUMO
Epidermal keratinocytes (EK) are exposed to multiple inflammatory stimuli and paracrine factors secreted by various dermal cells (lymphocytes, mast-cells, macrophages, fibroblasts) during wounding, cutaneous allergy and infections. We have previously demonstrated that following stimulation with interleukin-4 (IL-4) or interferon-gamma, human EK express the low affinity receptor for IgE (Fc epsilon RII/CD23) on their surface. In the present study, we showed that the ligation of CD23 by IgE/anti-IgE immune complexes or specific monoclonal antibody, induces a dose-dependent release of interleukin-6 and tumor necrosis factor-alpha from EK. CD23-ligation activates the nitric oxide-dependent pathway, as demonstrated by the high levels of nitrites released in cell supernatants, and the accumulation of intracellular cyclic nucleotides in EK. These second messengers are required for IgE-dependent stimulation of cytokine production by these cells, as this is completely abolished by cAMP or NO synthase antagonists. Human epithelial keratinocytes may thus participate in IgE-mediated immune responses, through their ability to express functional CD23 antigen.
Assuntos
AMP Cíclico/fisiologia , Imunoglobulina E/fisiologia , Queratinócitos/fisiologia , Óxido Nítrico/fisiologia , Receptores de IgE/fisiologia , Aminoácido Oxirredutases/antagonistas & inibidores , Arginina/análogos & derivados , Arginina/farmacologia , Bucladesina/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , GMP Cíclico/fisiologia , Células Epidérmicas , Humanos , Interleucina-6/metabolismo , Queratinócitos/metabolismo , Masculino , Óxido Nítrico Sintase , Receptores de IgE/biossíntese , Transdução de Sinais , Tionucleotídeos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , ômega-N-MetilargininaRESUMO
Acute myelogenous leukemia (AML) cells express CD23 surface antigen after in vitro treatment with various cytokines, including interleukin-4 (IL-4) and interferon gamma. Subsequent ligation of CD23 by specific monoclonal antibody (MoAb) induces substantial morphologic and functional modifications in these cells. In the present study, we investigated the role of CD23 in the proliferation and the maturation of leukemic cells from AML patients or the U937 cell line. CD23+ cell treatment with CD23 MoAb inhibited the proliferation of leukemic cells. This correlated with their terminal differentiation after 7 to 9 days incubation because they (1) definitively lost their growth capacity; (2) adhered to culture flasks and became monocyte/macrophage-like; and (3) expressed mature monocyte markers including nonspecific esterases. Intracellular mechanism of this antitumoral effect was then analyzed in U937 cells. Induction of high-density surface CD23 expression by IL-4 or granulocyte-macrophage colony-stimulating factor coincided with a transient decrease of U937 cell proliferation. CD23 ligation during this low-proliferative phase induced a rapid activation of L-arginine-dependent pathway and the intracellular accumulation of cyclic guanosine monophosphate and cyclic adenosine monophosphate (cAMP). Induction of these early messengers was followed by the activation of nuclear factor-kB transcription factor and the modulation of proto-oncogene expression by U937 cells. Whereas U937 cell treatment with IL-4 decreased c-fos/c-jun expression, CD23 MoAb reinduced c-fos/c-jun and promoted the expression of cell maturation-associated proto-oncogenes junB and c-fms, during the first 24 hours. Both IL-4 and CD23 MoAb downregulated the expression of c-myb. CD23 ligation also induced the production of TNF alpha by U937 cells. Inhibitors of cAMP and nitric oxide reversed CD23-mediated modification in U937 cells. These data evidence the ability of CD23 surface antigen to mediate terminal differentiation of early leukemic myelomonocytic cells.