RESUMO
Interleukin-5 is produced by a number of cell types, and is responsible for the maturation and release of eosinophils in the bone marrow. In humans, interleukin-5 is a very selective cytokine as a result of the restricted expression of the interleukin-5 receptor on eosinophils and basophils. Eosinophils are a prominent feature in the pulmonary inflammation that is associated with allergic airway diseases, suggesting that inhibition of interleukin-5 is a viable treatment. The present review addresses the data that relate interleukin-5 to pulmonary inflammation and function in animal models, and the use of neutralizing anti-interleukin-5 monoclonal antibodies for the treatment of asthma in humans.
Assuntos
Asma/imunologia , Eosinofilia/imunologia , Interleucina-5/imunologia , Células Th2/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Asma/terapia , HumanosRESUMO
The postjunctional alpha(2)-adrenoceptor-mediated contractility was characterized in human saphenous vein derived from coronary artery bypass graft surgery. Human saphenous vein contracted to alpha(2)-adrenoceptor selective agonists BHT-920 (5,6,7,8-Tetrahydro-6-(2-propenyl)-4H-thiazolo[4,5-d]azepin-2-amine dihydrochloride; pD(2)=6.7+/-0.1) and UK 14,304 (5-Bromo-6-(2-imidazolin-2-ylamino)quinoxaline; pD(2)=7.2+/-0.1). BHT-920-induced contractions were inhibited by the alpha(2)-adrenoceptor antagonist yohimbine (17-Hydroxy-yohimban-16-carboxylic acid methyl ester hydrochloride; pA(2)=8.7+/-0.5), but not by the alpha(1)-adrenoceptor antagonist prazosin (1-[4-Amino-6,7-dimethoxy-2-quinazolinyl]-4-[2-furanylcarbonyl]-piperazine hydrochloride; 300 nM). In contrast, prazosin (pK(b)=7.9+/-0.2) potently antagonized contractions elicited by the alpha(1)-adrenoceptor agonist phenylephrine ((R)-3-Hydroxy-alpha-[(methylamino)methyl] benzenemethanol hydrochloride; pD(2)=4.9+/-0.1), indicating that both alpha(2)- and alpha(1)-adrenoceptor evoke human saphenous vein contractions. Functional antagonist activity estimates (pA(2) or pK(b)) obtained for the alpha-adrenoceptor antagonists ARC 239 (2-[2-(4-(2-Methoxyphenyl)piperazin-1-yl)ethyl]-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione dihydrochloride), WB 4101 (2-(2,6-Dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride) and HV 723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy) ethyl)amino)propyl)benzeneacetonitrile) against BHT-920-induced human saphenous vein contractions were 7.0+/-0.6, 8.3+/-0.6 and 7.7+/-0.3, respectively. The alpha(2)-adrenoceptor subtype affinities (pK(i)) obtained in recombinant human alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptor competition binding assays were 8.6, 8.3 and 8.6 for yohimbine; 6.3, 8.4 and 7.0 for ARC 239; 8.4, 7.5 and 8.4 for WB 4101 and 7.5, 7.4 and 7.9 for HV 723, respectively. Taken together, the binding and functional antagonist activity estimates obtained in these investigations indicate that alpha(2C)-adrenoceptor is the predominant postjunctional alpha(2)-adrenoceptor subtype in human saphenous vein.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Veia Safena/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Idoso , Idoso de 80 Anos ou mais , Animais , Azepinas/farmacologia , Tartarato de Brimonidina , Células CHO , Cricetinae , Dioxanos/farmacologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Quinoxalinas/farmacologia , Receptores Adrenérgicos alfa 2/fisiologia , Veia Safena/fisiologia , Vasoconstrição/fisiologia , Ioimbina/farmacologiaRESUMO
Here we determine for norepinephrine, (5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline) (UK14,304), 5,6,7,8-tetrahydro-6-(2-propenyl)-4H-thiazolo[4,5-d]azepin-2-amine dihydrochloride (BHT-920), (2-[3-hydroxy-2,6-dimethyl-4-t-butylbenzyl]-2-imidazoline) (oxymetazoline), and ((R)-3-Hydroxy-alpha-[(methylamino)methyl]-benzenemethanol hydrochloride) (phenylephrine), affinities using a radiolabeled agonist and antagonist, and potency and efficacy values in membrane [(35)S]guanosine-5'-O-(3-thiotriphosphate) ([(35)S]GTP gamma S) binding and cAMP cellular inhibition assays, in Chinese hamster ovary cells (CHO-K1) expressing the human alpha(2c)-adrenoceptor. These cells express a high ratio of receptor to G-protein because each agonist, but not several antagonists, displaced [(3)H]UK14,304 with higher affinity than [(3)H]rauwolscine. The rank order of potency of high affinity K(i) and EC(50) in both functional assays was norepinephrine > or =UK14,304>BHT-920>oxymetazoline>phenylephrine. The receptor reserve of G-protein activation and cAMP responses was measured with the irreversible antagonist, benextramine; K(A) values of norepinephrine or UK14,304 were similar (289, 271 or 150, 163 nM, respectively). A 20-fold greater receptor occupancy was required for agonist-induced half-maximal [(35)S]GTP gamma S binding compared to cAMP inhibition, indicating significant signal amplification in cells. Therefore, the G-protein activation assay is better at distinguishing full and partial agonists.
Assuntos
AMP Cíclico , Guanosina 5'-O-(3-Tiotrifosfato) , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Ligação Competitiva/efeitos dos fármacos , Células CHO , Cricetinae , AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Cinética , Transdução de Sinais/efeitos dos fármacos , Radioisótopos de Enxofre , Ioimbina/farmacologiaRESUMO
Human interleukin (IL)-5 gene transcription is regulated by several transcription factor binding sites, including CLE 0, GATA, and a region from position -123 to -92 known as response element (RE)-II. By expression cloning, a partial protein was identified that bound to concatamers of RE-II. Recombinant protein derived from this initial complementary DNA (cDNA) encoding the partial protein specifically bound to RE-II-containing oligonucleotides in electromobility shift assays. The complete sequence (3,649 bp) was determined by 5' rapid amplification of cDNA ends and comparisons to existing ESTs, and found to be identical to the 3' half of Wolf-Hirschhorn syndrome candidate 1, (WHSC1; also known as Multiple Myeloma SET domain [MMSET]). The full-length protein contains an SET domain and two plant homeodomain-type zinc fingers. Transcription initiation of RE-II binding protein (RE-IIBP) messenger RNA (mRNA) uniquely occurred within the middle of WHSC1 near a region that exhibits complex mRNA splicing. RE-IIBP reactive polyclonal antisera identified proteins in human T-cell nuclear protein extracts of 62 and 66 kD that were consistent with the length of the longest open reading frame in RE-IIBP. In contrast, WHSC1 is predicted to encode a protein of 136 kD. In activated human Jurkat and murine D10.G4.1 T cells, expression of full-length and truncated forms of RE-IIBP repressed RE-II promoter activity of a 5X-RE-II luciferase reporter construct by as much as 75%. In addition, RE-IIBP expressed in activated D10.G4.1 T cells inhibited endogenous murine IL-5 production. The repressor activity of RE-IIBP is consistent with the presence of an SET domain that is found in other proteins that act as gene silencers.
Assuntos
Proteínas de Transporte/genética , Proteínas de Grupo de Alta Mobilidade/genética , Interleucina-5/genética , RNA Mensageiro/genética , Transcrição Gênica/genética , Processamento Alternativo/genética , Animais , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Linhagem Celular , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/farmacologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Histona-Lisina N-Metiltransferase , Humanos , Interleucina-5/antagonistas & inibidores , Interleucina-5/biossíntese , Íntrons/genética , Camundongos , Iniciação Traducional da Cadeia Peptídica/genética , Estrutura Terciária de Proteína/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/farmacologia , Análise de Sequência de DNA , Transcrição Gênica/efeitos dos fármacos , Dedos de ZincoRESUMO
A novel pharmacological study of CCR3 receptor reserve in a CCR3-transfected cell (CREM3) and human eosinophils was done; functional responses measured were increases in intracellular calcium and chemotaxis. Eotaxin, eotaxin-2, monocyte chemoattractant protein-4 (MCP-4), RANTES, and MCP-3 induced similar maximal eosinophil chemotaxis, whereas MCP-3 and RANTES induced submaximal calcium responses in eosinophils compared to eotaxin, MCP-4, and eotaxin-2. This suggested a receptor reserve in the chemotaxis response. Receptor reserve was quantitated for eotaxin. Occupancy of all CCR3 receptors was required for a maximal calcium response in both CREM3 and eosinophils (reserve = 1.0 or 0.17, respectively); the stimulus-calcium response relationship was linear, indicating no receptor reserve. In contrast, in eosinophils a large receptor reserve (6.5) was found for chemotaxis, where occupancy of 15% receptors drove half-maximal responses. These studies indicate that CCR3 interacts with G-proteins that are poorly coupled to the calcium response, whereas coupling efficiency and/or amplification to the chemotaxis apparatus in human eosinophils is significantly greater.
Assuntos
Quimiocinas CC , Eosinófilos/metabolismo , Receptores de Quimiocinas/agonistas , Receptores de Quimiocinas/metabolismo , Animais , Ligação Competitiva , Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular , Células Cultivadas , Quimiocina CCL11 , Quimiocina CCL5/metabolismo , Quimiocina CCL5/farmacologia , Fatores Quimiotáticos de Eosinófilos/metabolismo , Fatores Quimiotáticos de Eosinófilos/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Citocinas/metabolismo , Citocinas/farmacologia , Relação Dose-Resposta a Droga , Eosinófilos/citologia , Eosinófilos/efeitos dos fármacos , Humanos , Ligantes , Proteínas Quimioatraentes de Monócitos/metabolismo , Proteínas Quimioatraentes de Monócitos/farmacologia , Ratos , Receptores CCR3 , Receptores de Quimiocinas/genética , Termodinâmica , TransfecçãoRESUMO
Identification of chemokine receptors and their associated ligands is crucial to the understanding of most immune reactions. Three human chemokines [I-309, thymus and activation-regulated chemokine (TARC) and macrophage inflammatory protein-1beta (MIP-1beta)] have been reported to be ligands for CC-chemokine receptor 8 (CCR8). In this report, we present evidence that TARC and MIP-1beta did not bind to or induce chemotaxis through CCR8 on a stable transfected cell line (1D-21) and did not bind to CCR8 on in vitro differentiated human CD4(+) Th(2) cell cultures. Also, I-309-dependent calcium mobilization in 1D-21 cells and in Th(2) cells was desensitized by I-309 but not by MIP-1beta or TARC. These results provide strong evidence that, at physiologically relevant concentrations, I-309 is the only known human ligand for CCR8. These data also provide a framework for suggesting minimum requirements for the assignment of chemokine receptor-ligand pairs.
Assuntos
Quimiocinas CC/metabolismo , Proteínas Inflamatórias de Macrófagos/metabolismo , Receptores de Quimiocinas/metabolismo , Antígenos CD4/biossíntese , Antígenos CD4/genética , Cálcio/metabolismo , Células Cultivadas , Quimiocina CCL17 , Quimiocina CCL4 , Quimiocinas CC/imunologia , Quimiotaxia de Leucócito/imunologia , DNA/genética , Humanos , Ligantes , Proteínas Inflamatórias de Macrófagos/imunologia , Receptores CCR8 , Receptores de Quimiocinas/biossíntese , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/imunologia , Células Th2/imunologia , Células Th2/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
We compared the effects of cyclosporin A (CSA) and a macrotetrolide antibiotic, dinactin, on human T-cell proliferation and cytokine production induced by stimulation of the T-cell receptor alone (monoclonal antibody [mAb] directed against CD3) or in combination with costimulatory signals (mAbs directed against CD3 and CD28). These agents were also examined in a murine model of interleukin (IL)-5-mediated pulmonary inflammation. Dinactin inhibited T-cell proliferation induced by IL-2, by mAb to CD3, and by mAbs to CD3 plus alpha-CD28 with identical dose-response curves (IC50 = 10-20 ng/ml). Dinactin inhibited cytokine production with IC50 values of 10 ng/ml for IL-4 and IL-5 and 30 or 60 ng/ml for interferon-gamma or IL-2, respectively. Unlike CSA, exogenous IL-2 did not alter the dinactin-mediated effects on T cells, and nuclear run-on and steady-state messenger RNA (mRNA) analysis showed that dinactin inhibited cytokine production through a post-transcriptional mechanism. CSA selectively blocked T-cell receptor-induced T-cell proliferation and cytokine production (IC50 = 10 ng/ml). Under costimulatory conditions, IL-5 synthesis was only minimally inhibited by high concentrations of CSA, and at CSA concentrations of less than 125 ng/ml, IL-5 was significantly increased above control values. Dinactin and CSA reduced pulmonary eosinophilia when administered within 1 d of airway antigen challenge. Of the cytokine mRNAs examined in the lungs of CSA-pretreated, antigen-challenged mice, IL-5 mRNA levels were the least reduced, paralleling the resistance of IL-5 to CSA observed in vitro and suggesting a role for CD28 in the in vivo induction of IL-5.
Assuntos
Antibacterianos/farmacologia , Linfócitos T CD4-Positivos/imunologia , Ciclosporina/farmacologia , Hipersensibilidade/imunologia , Interleucina-5/biossíntese , Macrolídeos , Hipersensibilidade Respiratória/imunologia , Animais , Antígenos CD28/imunologia , Complexo CD3/imunologia , Citocinas/biossíntese , Relação Dose-Resposta a Droga , Humanos , Ativação Linfocitária , Camundongos , RNA Mensageiro/análiseRESUMO
The maturation of eosinophils in bone marrow, their migration to pulmonary tissue, and their subsequent degranulation and release of toxic granule proteins contributes to the pathophysiology observed in asthma. Interleukin-5 (IL-5) is essential for these processes to occur. Therefore, much emphasis has been placed on attempts to inhibit the production or activity of IL-5 in order to attenuate the inflammatory aspect of asthma. In this report, the immunological consequences of long-term exposure to an antibody recognizing IL-5 (TRFK-5) were studied in a murine pulmonary inflammation model. A single dose of TRFK-5 (1 mg/ kg, intraperitoneally) reversibly inhibited antigen-dependent lung eosinophilia in mice for at least 12 wk and inhibited the release of eosinophils from bone marrow for at least 8 wk. Normal responses to aerosol challenge were attained after 24 wk. In mice treated acutely with antibody (2 h before challenge), 50% inhibition of pulmonary eosinophilia occurred when 0. 06 mg/kg TRFK-5 was administered (intraperitoneally; ED50), resulting in 230 ng/ml (IC50) in serum. In mice treated with one dose of TRFK-5 (1 mg/kg) and rested before challenge, the antibody exhibited a half-life of 2.4 wk. After 18 to 19 wk, antigen challenge-induced eosinophilia was inhibited by 50% and serum levels of TRFK-5 were 25 ng/ml. TRFK-5 remaining in mice 8 wk after a single injection of TRFK-5 was sufficient to inhibit at least 50% of the eosinophilia induced in blood 3 h after injection of recombinant murine IL-5 (10 microg/kg, intravenously). To assess the biologic effect of long-term exposure of mice to antibody, several parameters of immune-cell function were measured. Throughout the extended period of activity of TRFK-5 (>/= 12 wk) there were no gross effects on antigen-dependent increases in T-cell recruitment into bronchoalveolar fluid (BALF), in IL-4 and IL-5 steady-state mRNA levels in lung tissue, or in immunoglobulin E (IgE) and IgG levels in serum. There was a small increase in IL-5 steady-state mRNA production in TRFK-5-treated mice after 2 h or 2 wk, but this was not observed at other times examined. In untreated mice, IL-5 steady-state mRNA production in response to antigen challenge decreased > 6-fold with age, although at all time points there was an increase in mRNA levels following challenge. Therefore, at later times, 25 ng/ml rather than 230 ng/ml of TRFK-5 inhibited BALF eosinophilia, probably because of reduced IL-5 levels. Twenty-four weeks after treatment with TRFK-5, when challenge-induced eosinophilia was restored, there was an excess of CD4(+) T cells in BALF from challenged mice. However, these T cells had no measurable effects on other responses to challenge, including cytokine production, B-cell accumulation, and immunoglobulin production in serum. Thus, the biologic duration of TRFK-5 was several months, and its activity was due to the presence of antibody above a therapeutic threshold rather than to any profound effect on the immune system.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Interleucina-5/imunologia , Pneumonia/terapia , Animais , Anticorpos Monoclonais/sangue , Linfócitos B/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Modelos Animais de Doenças , Eosinofilia/complicações , Eosinofilia/terapia , Interleucina-4/biossíntese , Interleucina-5/biossíntese , Interleucina-5/genética , Masculino , Camundongos , Pneumonia/sangue , Pneumonia/complicações , RNA Mensageiro/sangue , Linfócitos T/imunologiaRESUMO
The proximal mouse IL-5 promoter was examined using a mouse TH2 clone stimulated through the T cell receptor using anti-CD3 monoclonal antibody. DNase I protection defined four protein binding regions [IL-5RE-A, -69/-45; -B, (-90/-76); -C, (-154/-130); and -D (-176/-157)]. Stimulation-dependent binding, which was seen in the IL-5RE-B, -D regions and the 5' end of tIL-5RE-A, did not require new protein synthesis inhibitor during cell activation. EMSA using probes targeted to the IL-5RE-B, -C, -D regions demonstrated the multimeric nature of the bound proteins. By transfection analysis using a series of truncated IL-5 promoter-luciferase constructs, IL-5RE-C and -D contributed little to constitutive or inducible activity. The CLE0 site in the IL-5RE-A region contributed to full transcriptional activity but was not sufficient to mediate full activity. Full stimulation-dependent activity required the IL-5RE-B region and/or the GATA site (-70/-60).
Assuntos
Interleucina-5/genética , Regiões Promotoras Genéticas , Células Th2/imunologia , Animais , Sequência de Bases , Sítios de Ligação , Núcleo Celular/metabolismo , Células Clonais , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I , Luciferases/biossíntese , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/biossínteseRESUMO
Interleukin-5 (IL-5) transcriptional activation and mRNA stability were investigated in a human TH0 T-cell clone (SP-B21) and in nonclonal CD4 TH2 cells, differentiated in vitro from peripheral blood T cells. Cells were stimulated with alpha-CD3 monoclonal antibody (mAb) with and without alpha-CD28 mAb. Comparison to other cytokine genes revealed aspects of mRNA regulation unique to IL-5. The half-life (t1/2) of IL-5 mRNA, determined by addition of actinomycin D (ActinoD) or cyclosporin A (CSA) was longer (by >= 2 h) than that of IL-2, IL-3, IL-4, interferon-gamma, or granulocyte/macrophage colony-stimulating factor. With the exception of IL-5, t1/2 values were significantly shorter with CSA as the transcriptional inhibitor than with ActinoD. The t1/2 value of IL-5 mRNA, but not the other cytokine transcripts, determined with either ActinoD or CSA, was longer than predicted from the kinetics of steady-state mRNA decline. Co-stimulation of both cell types with alpha-CD28 mAb increased the stability of cytokine transcripts weakly, and IL-5 remained the most stable transcript. Thus, the degradation pathway that targets IL-5 is distinct from the other cytokine transcripts measured and involves proteins whose transcription is blocked by ActinoD and CSA. From examination of the levels of transcription initiation (nuclear run-on assay) and steady-state mRNA attained in cultures stimulated in the presence of the protein synthesis inhibitor, cycloheximide, only IL-5 transcription initiation had an absolute dependency on new protein synthesis.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Regulação da Expressão Gênica/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Interferon gama/genética , Interleucinas/genética , Anticorpos Monoclonais/farmacologia , Antígenos CD28/imunologia , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/química , Linfócitos T CD4-Positivos/citologia , Células Cultivadas , Ciclosporina/farmacologia , Dactinomicina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunossupressores/farmacologia , Interleucina-2/genética , Interleucina-3/genética , Interleucina-4/genética , Interleucina-5/biossíntese , Interleucina-5/genética , Cinética , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA Mensageiro/metabolismo , Transcrição Gênica/imunologiaRESUMO
Asthma is characterized by acute episodes of nonspecific airway hyperreactivity and chronic pulmonary inflammation exacerbated by stimuli including allergen exposure. In order to reproduce the physiologic and immunologic responses that occur in asthmatic patients, we have characterized a model of antigen-induced inflammation in which allergic mice (B6D2F1) that had been challenged once with aerosolized ovalbumin and had developed a pulmonary cellular infiltrate were rechallenged 1 wk later. Pulmonary inflammation in rechallenged mice was substantially greater than that in single-challenged mice. Eosinophils and activated-memory T cells (CD44+, CD45RBlo) in bronchoalveolar lavage (BAL) fluid accumulated to higher levels and with faster kinetics in response to the second challenge than in response to the first challenge. Eosinophils in lung tissue also accumulated to higher levels but with similar kinetics in response to the second challenge than in response to the first challenge. Similarly, interleukin (IL)-4 and IL-5 steady-state mRNA levels in lung tissue increased after the second challenge and were higher than those measured after a single challenge. Furthermore, treatment of mice with an anti-IL-5 monoclonal antibody 2 h prior to rechallenge inhibited antigen induced eosinophil accumulation in the lungs. In mice challenged twice, peak in vivo bronchoconstrictor responsiveness to acetylcholine was increased following the second challenge compared with that observed following the initial challenge. In contrast, ex vivo tracheal smooth muscle contractile responsiveness to acetylcholine was not altered. Although mucus accumulation and epithelial damage in pulmonary tissue were evident in mice challenged twice, these parameters were slightly reduced compared with those seen at similar times in mice challenged once. Therefore, although these mice exhibit only slight bronchial epithelial damage, the presence of significant inflammation and airway hyperreactivity to acetylcholine as well as slightly increased baseline reactivity demonstrate important similarities with the pathophysiology of asthma.
Assuntos
Asma/imunologia , Testes de Provocação Brônquica , Citocinas/imunologia , Eosinófilos/imunologia , Inflamação/imunologia , Pulmão/imunologia , Ovalbumina/administração & dosagem , Linfócitos T/imunologia , Administração por Inalação , Animais , Eosinófilos/patologia , Pulmão/patologia , Camundongos , Linfócitos T/patologiaRESUMO
This study was conducted to directly compare the in vitro efficacy and potency of several glucocorticoids in inhibiting T-cell cytokine production. The glucocorticoids tested were fluticasone propionate, budesonide, triamcinolone acetonide, and beclomethasone dipropionate, which are currently inhaled therapies for the treatment of allergic airway disease. Also used were betamethasone phosphate and the newly developed mometasone furorate. With a novel cell culture system, purified peripheral blood CD4+ T cells from normal donors were stimulated with immobilized anti-CD3 and soluble anti-CD28 monoclonal antibodies to induce high levels of IL-4, IL-5, and interferon-gamma. By cell sorting, it was found that the IL-5 produced originated from memory cells, whereas both memory and naive cells produced interferon-gamma. Mometasone and fluticasone inhibited IL-5 and IL-4 similarly (mometasone IL-5 inhibitory concentration of 50% = 0.27 +/- 0.1 nmol/L and IL-4 = 0.19 +/- 0.08 nmol/L). For both cytokines, the results indicate that mometasone and fluticasone were more potent than beclomethasone, triamcinolone, budesonide, and betamethasone. Of clinical importance is the finding that all steroids demonstrated less efficacy versus interferon-gamma than IL-4 and IL-5. Glucocorticoid reduction of Th2 cytokines with lesser effects on interferon-gamma would serve to reverse the exaggerated Th2 response that contributes to pathophysiology observed in allergic disease. Therefore the use of topically active glucocorticoids with low systemic bioactivity for the treatment of allergic inflammation may be particularly effective in modulating the cytokine activity that is an important component of the allergic response.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Interferon gama/biossíntese , Interleucina-4/biossíntese , Interleucina-5/biossíntese , Separação Celular , Células Cultivadas , Depressão Química , Citometria de Fluxo , Humanos , Interferon gama/metabolismo , Interleucina-4/metabolismo , Interleucina-5/metabolismo , Estimulação Química , Subpopulações de Linfócitos T/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Células Th2/metabolismoRESUMO
This study identifies three regions of the human interleukin (IL)-5 promoter involved in binding nuclear factors from activated T cells. DNase I footprinting and mobility shift assays with nuclear proteins from the human T cell clone, SP-B21, demonstrated protein interactions with each of these response elements (REs), located between positions -79 and -45 (RE-I), -123 and -92 (RE-II), and -170 and -130 (RE-III). Two of these regions, RE-II and RE-III, have not previously been described to regulate IL-5 expression in T cells. The RE-II site was shown to be critical for inducible IL-5 promoter activity in transient transfection assays in D10.G4.1 T cells, while the RE-III site functions as a negative regulatory element. The activity of the RE-II site was specifically inhibited by cyclosporin A, and transfection assays with IL-5 constructs containing mutations in the RE-II site showed greatly reduced reporter gene activity. We have defined the sequence involved in stimulation-dependent transcription and have identified constitutive as well as inducible DNA-binding protein complexes that bind to RE-II. Antibodies against at least two members of the nuclear factor of activated T cells (NFAT) family of transcription factors are capable of binding to the IL-5 RE-II complexes, although they can be distinguished from previously identified NFAT-specific complexes by several characteristics.
Assuntos
Regulação da Expressão Gênica , Interleucina-5/genética , Proteínas Nucleares , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Sítios de Ligação , Ciclosporina/farmacologia , DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Humanos , Interleucina-2/genética , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição NFATC , Regiões Promotoras GenéticasRESUMO
Pulmonary inflammation is characterized by the accumulation of eosinophils and other leukocytes in the lungs of individuals challenged with antigen. Cytokines released by the Th2 lymphocyte subset, especially interleukin-4 (IL-4) and interleukin-5 (IL-5), are also present and thought to play an important role in this process. Previously, we used a model of aerosolized antigen challenge of sensitized mice to show that T cells were necessary for the accumulation of eosinophils and the production of cytokine steady-state messenger ribonucleic acid (mRNA). T cells were isolated from lung tissue at a time (4 h) when high levels of IL-4 and IL-5 mRNAs had accumulated, and from bronchoalveolar lavage fluid (BALF) and lung tissue at a later time (24 h), when inflammation could be detected by lavage. Lung-derived lymphocytes from sensitized challenged mice consisted of approximately 40% Thyl+ T cells (20% CD4+, 13% CD8+, and 6% CD4+/CD8+) and 30% B220+ B cells. Both BALF- and lung-derived T lymphocytes exhibited a similar activated/memory phenotype (CD44+ CD45RBlo), although lung tissue also contained less differentiated cells (CD44+ CD45RBhi). Thyl+ BALF cells isolated by magnetic bead-mediated separation accounted for approximately 88% of the IL-5 mRNA, 21% of the interferon-gamma (IFN-gamma) mRNA, and < 2% of the IL-4 mRNA detected in unseparated samples by reverse transcriptase-polymerase chain reaction (RT-PCR). Thyl+ T cells from lung tissue accounted for approximately 98% and 89% of IL-5 mRNA, 56% and 80% of IFN-gamma mRNA, and 23% and 40% of IL-4 mRNA at 4 h and 24 h after challenge, respectively. These experiments demonstrate that isolated T cells from BALF and lung are responsible for most of the IL-5 mRNA, but not all of the IFN-gamma or IL-4 mRNAs, detected in this model. These results are consistent with human studies indicating T cells as the major source of IL-5 mRNA in the lungs of asthmatic patients.
Assuntos
Hipersensibilidade/imunologia , Interleucina-4/genética , Interleucina-5/genética , Linfócitos T/fisiologia , Animais , Antígenos/farmacologia , Linfócitos B/fisiologia , Líquido da Lavagem Broncoalveolar/citologia , Expressão Gênica/imunologia , Hipersensibilidade/genética , Hipersensibilidade/metabolismo , Memória Imunológica/fisiologia , Imunofenotipagem , Interferon gama/genética , Pulmão/citologia , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Linfócitos T/química , Antígenos Thy-1/análiseRESUMO
Cis-acting regions in the 5'-flank of the mouse interleukin 5 (IL-5) gene involved in the specific and inducible regulation of IL-5 transcription in an untransformed mouse T cell clone, D10.G4.1, have been identified. Transient transfection assays with a series of deletion IL-5 promoter reporter constructs indicate that multiple regulatory elements in the 5'-flanking region of the IL-5 promoter play a role in regulating IL-5 transcription in Th2 cells. Negatively acting elements, NRE I and NRE II, map to the regions between positions -431 and -392 and positions -300 and -261. A positive regulatory element has been mapped to the region between positions -224 and -81. The activity of these elements is dependent on activation of the cells. A 40-bp sequence within this region, termed the IL-5 PRE, has been shown to bind at least two specific nuclear protein complexes from unstimulated and stimulated D10.G4.1 cells. An additional protein complex specific for this site has been identified in nuclear fractions from cells stimulated in the presence of the protein synthesis inhibitor, cycloheximide. Proteins that bind to these elements are likely to be important inducible and specific factors essential for control of IL-5 transcription in response to T cell receptor-mediated signaling events.
Assuntos
Regulação da Expressão Gênica , Interleucina-5/biossíntese , Interleucina-5/genética , Camundongos/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Sequência de Bases , Northern Blotting , Células Clonais , Clonagem Molecular , Primers do DNA , Luciferases/biossíntese , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos , Proteínas Recombinantes/biossíntese , Sequências Reguladoras de Ácido Nucleico , Linfócitos T , TransfecçãoAssuntos
Hipersensibilidade/terapia , Interferon gama/uso terapêutico , Interleucina-4/imunologia , Interleucina-5/imunologia , Eosinofilia Pulmonar/terapia , Animais , Medula Óssea/imunologia , Imunoterapia , Interferons , Camundongos , Camundongos Endogâmicos , Ovalbumina/imunologia , Eosinofilia Pulmonar/patologia , Proteínas RecombinantesRESUMO
In a murine model of pulmonary inflammation, aerosolized antigen challenge of sensitized B6D2F1 mice leads to eosinophil accumulation within the lungs. Little is known of the role of T cells and their cytokine products in these allergic animals. In this study, we show that T cells migrate into the lungs in response to antigen challenge and are necessary for local production of cytokines (IL-4 and IL-5) important in B and T cell development as well as eosinophil activation and differentiation. Flow cytometry revealed an increase in the percentage of Thy1+ T cells but not in B220+ B cells in bronchoalveolar lavage fluid after challenge when compared to unchallenged mice. Although there was an increase in both T cell subsets, there were twice as many CD4+ cells as CD8+ cells at 24 hr and after 48 hr the CD4+ subset predominated. The CD4+ T lymphocytes were CD44+ CD45RBlo indicating an activated/memory phenotype and tracheobroncheal lymph node cells obtained from challenged mice proliferated in a dose-dependent manner in response to antigen stimulation in vitro. Reverse transcriptase-polymerase chain reaction analysis of lung tissue-derived RNA indicated an increase in Th2-like cytokines. IL-4 and IL-5 steady-state mRNAs were at peak levels 6 hr after challenge, while no consistent increase was found for IFN-gamma mRNA levels. Treatment with the glucocorticoid betamethasone just prior to challenge reduced the levels of cytokine mRNA as well as the eosinophil influx. In vivo depletion of T cells from sensitized mice reduced pulmonary eosinophilia as well as the expression of IL-4, IL-5, and IFN-gamma steady-state mRNAs in the lungs of sensitized and challenged mice. These results indicate that T cells migrating into the lungs of mice after antigen challenge play an important role in the production of Th2-like cytokines and the accumulation of eosinophils in bronchial fluids.
Assuntos
Citocinas/metabolismo , Eosinófilos/citologia , Hipersensibilidade Respiratória/imunologia , Linfócitos T/fisiologia , Células Th2/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Antígenos/administração & dosagem , Líquido da Lavagem Broncoalveolar/citologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Proteínas de Transporte/genética , Receptores de Hialuronatos , Memória Imunológica , Interleucina-4/metabolismo , Interleucina-5/metabolismo , Isoanticorpos/farmacologia , Pulmão/imunologia , Pulmão/patologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos , Fenótipo , Pneumonia/imunologia , Receptores de Superfície Celular/genética , Receptores de Retorno de Linfócitos/genética , Hipersensibilidade Respiratória/patologia , Linfócitos T/imunologiaRESUMO
Mast cells are important effector cells in IgE-mediated acute allergic reactions. Mast cells also produce cytokines such as interleukin (IL)-3, IL-4, IL-5, tumor necrosis factor (TNF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) that regulate the function of eosinophils and the development of a late-phase inflammatory response to antigen challenge. To evaluate the role of mast cells on the development of IgE-mediated allergic pulmonary eosinophilia in vivo, we compared the eosinophil infiltration into lungs of mast cell deficient mice (WBB6F1/J-W/Wv) with their congenic normal littermates (W/W+). Mice were sensitized with alum-precipitated ovalbumin and challenged with aerosolized ovalbumin on day 12 after sensitization. Bronchoalveolar lavage (BAL) fluid, lung tissue biopsies, and blood samples were collected after ovalbumin challenge. Eosinophil numbers in the BAL and lung tissue, lung eosinophil peroxidase (EPO) activity and serum levels of IgE and IgG1 were measured. In sensitized W/W+ mice, there were increased numbers of eosinophils in the BAL fluid and lung tissue, and EPO levels were increased after ovalbumin challenge. Ovalbumin challenge of sensitized mast-cell-deficient mice produced fewer numbers of eosinophils in the BAL fluid and lungs, and EPO levels were also reduced compared with their challenged congenic littermates. On the other hand, levels of serum IgE and IgG1 were not different between W/Wv mice and their congenic littermates.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Eosinófilos/imunologia , Hipersensibilidade/imunologia , Mastócitos/imunologia , Pneumonia/imunologia , Animais , Células da Medula Óssea , Testes de Provocação Brônquica , Líquido da Lavagem Broncoalveolar/citologia , Movimento Celular , Peroxidase de Eosinófilo , Imunização Passiva , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Masculino , Camundongos , Camundongos Mutantes , Ovalbumina , Peroxidases/metabolismoRESUMO
Pulmonary inflammation with eosinophil (EOs) infiltration is a prominent feature of allergic respiratory diseases such as asthma. In order to study the cellular response during the disease development, an animal model of IgE-mediated pulmonary inflammation with characteristic eosinophilia is needed. We developed a method for inducing severe pulmonary eosinophilia in the mouse and also studied the numbers of EOs in blood and bone marrow and the response to corticosteroid treatment. Animals were sensitized with alum-precipitated ovalbumin (OVA) and challenged with aerosolized OVA 12 days later when serum IgE levels were significantly elevated. Four to eight hours after challenge there were moderate increases in the number of EOs in the bone marrow and peripheral blood, but only a few EOs were observed in the lung tissue and in bronchoalveolar lavage (BAL) fluid. Twenty-four hours after challenge, there was a marked reduction of EOs in bone marrow, while the number of EOs peaked in the perivascular and peribronchial regions of the lung. Forty-eight hours after challenge, the highest number of EOs was found in the BAL fluid, making up > 80% of all cells in that compartment. The high levels of EOs in the lung tissue and BAL fluid lasted for 2-3 days and was followed by a more moderate but persistent eosinophilia for another 10 days. Nonsensitized animals showed no significant changes in the number of EOs in BAL fluid, lungs, blood or bone marrow. Histopathological evaluation also revealed epithelial damage, excessive mucus in the lumen and edema in the submucosa of the airways.(ABSTRACT TRUNCATED AT 250 WORDS)