RESUMEN

beta-Endorphin-like decapeptide immunorphin (SLTCLVKGFY), a selective agonist of non-opioid beta-endorphin receptor, was labeled with tritium to specific activity of 24 Ci/mmol. It was used for the detection and characterization of non-opioid beta-endorphin receptors on rat adrenal cortex membranes (Kd1 = 39.6 +/- 2.0 nM, Bmax1 = 40.7 +/- 2.3 pmol/mg protein; Kd2 = 0.25 +/- 0.01 micro M, Bmax2 = 187.8 +/- 9.4 pmol/mg protein). beta-Endorphin was found to inhibit the [3H]immunorphin specific binding to membranes (Ki = 70.0 +/- 9.2 nM); naloxone, [Met5]enkephalin, and alpha- and gamma-endorphins tested in parallel were inactive. Immunorphin at concentrations of 10(-9)-10(-6) M was found to inhibit the adenylate cyclase activity in adrenocortical membranes, while intramuscular injection of immunorphin at doses of 10-100 micro g/kg was found to reduce the secretion of 11-oxycorticosteroids from the adrenals to the bloodstream.

Asunto(s)

Corteza Suprarrenal/metabolismo , Receptores Opioides/metabolismo , 11-Hidroxicorticoesteroides/sangre , 11-Hidroxicorticoesteroides/metabolismo , Adenilil Ciclasas/metabolismo , Corteza Suprarrenal/citología , Corteza Suprarrenal/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Membrana Celular/metabolismo , Regiones Constantes de Inmunoglobulina , Cadenas gamma de Inmunoglobulina , Masculino , Oligopéptidos/antagonistas & inhibidores , Oligopéptidos/química , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Ratas , Ratas Wistar , Receptores Opioides/agonistas , Tritio , betaendorfina/farmacología

RESUMEN

Tritium-labeled selective agonist of non-opioid beta-endorphin receptor, the decapeptide immunorphine ([3H]SLTCLVKGFY) with specific activity of 24 Ci/mmol has been prepared. By its use, non-opioid beta-endorphin receptors were revealed and characterized on mouse peritoneal macrophages and rat myocardium, spleen, adrenal, and brain membranes. The non-opioid beta-endorphin receptor of macrophages has in addition to immunorphine (Kd of the [3H]immunorphine-receptor complex was 2.4 +/- 0.1 nM) and beta-endorphin (Ki of the [3H]immunorphine specific binding was 2.9 +/- 0.2 nM) a high affinity for Fc-fragment of human IgG1, pentarphine (VKGFY), cyclopentarphine [cyclo(VKGFY)], and [Pro3]pentarphine (VKPFY) (Ki values were 0.0060 +/- 0.0004, 2.7 +/- 0.2, 2.6 +/- 0.2, and 2.8 +/- 0.2 nM, respectively) and is insensitive to naloxone and [Met5]enkephalin (Ki > 100 microM). Treatment of macrophages with trypsin resulted in the loss of their ability for the specific binding of [3H]immunorphine. Values of the specific binding of 8.4 nM [3H]immunorphine to rat adrenal, spleen, myocardium, and brain membranes were determined to be 1146.0 +/- 44.7, 698.6 +/- 28.1, 279.1 +/- 15.4, and 172.2 +/- 1.8 fmol/mg protein, respectively. Unlabeled beta-endorphin, pentarphine, [Pro3]pentarphine, cyclopentarphine, cyclodipentarphine [cyclo(VKGFYVKGFY)], and Fc-fragment of IgG1 inhibited the binding of [3H]immunorphine to membranes from these organs. No specific binding of [3H]immunorphine to rat liver, lung, kidney, and intestine membranes was found.

Asunto(s)

Receptores Opioides/análisis , Secuencia de Aminoácidos , Animales , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/química , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Masculino , Membranas/citología , Membranas/metabolismo , Ratones , Datos de Secuencia Molecular , Morfina/química , Morfina/metabolismo , Morfina/farmacología , Oligopéptidos/química , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Ratas , Receptores Opioides/agonistas , Receptores Opioides/metabolismo

RESUMEN

Adrenocorticotropic hormone (ACTH)-like peptide immunocortin (IMC) VKKPGSSVKV, corresponding to the amino acid sequence 11-20 of the variable part of human immunoglobulin G (IgG) 1 heavy chain, at concentrations of 10(-9)-10(-6) I was found to increase the adenylate cyclase activity in adrenal cortex membranes, while intramuscular injection of immunocortin at doses of 10-100 microg/kg was found to stimulate the secretion of 11-oxycorticosteroids (CS) from the adrenals to the bloodstream. Immunocortin was labeled with tritium to specific activity of 22 Ci/mmol. Receptor binding studies revealed that [(3)H]immunocortin ([(3)H]IMC) bound with high affinity and specificity to ACTH receptors on rat adrenal cortex membranes (K(d)=2.1+/-0.2 nM, B(max)=1.1+/-0.1 pmol/mg protein).

Asunto(s)

11-Hidroxicorticoesteroides/metabolismo , Corteza Suprarrenal/metabolismo , Inmunoglobulina G/química , Fragmentos de Péptidos/química , Péptidos/química , Receptores de Corticotropina/metabolismo , Corteza Suprarrenal/efectos de los fármacos , Glándulas Suprarrenales/metabolismo , Secuencia de Aminoácidos , Animales , Unión Competitiva , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Inmunoglobulina G/metabolismo , Región Variable de Inmunoglobulina/química , Cinética , Datos de Secuencia Molecular , Unión Proteica , Ratas , Sensibilidad y Especificidad , Factores de Tiempo , Tritio/química

RESUMEN

We have synthesized two peptides, VKGFY and cyclo(VKGFY) (referred to as pentarphin (PNT) and cyclopentarphin (cPNT), respectively), and found that both peptides at 1 nM concentration increased the adhesion and spreading of murine peritoneal macrophages as well as their bactericidal activity in vitro, as shown by phagocytosis of Salmonella typhimurium virulent strain 415. PNT administered intraperitoneally at dose 20 microg/mouse on day 7, 3, and 1 prior to the isolation of macrophages also enhanced the macrophage adhesion and spreading. The receptor binding characteristics of PNT and cPNT were examined using 125I-labeled PNT. The binding of labeled PNT to peritoneal macrophages was high-affinity (K(d)=3.6 nM) and saturable. It was not inhibited by naloxone (NAL) or [Met(5)]enkephalin ([Met(5)]ENK) but completely inhibited by unlabeled cPNT (K(i)=2.6 nM), immunorphin (IMN, decapeptide SLTCLVKGFY, corresponding to the IgG heavy-chain sequence 364-373) (K(i)=3.2 nM) or beta-endorphin (beta-END) (K(i)=2.8 nM). Thus, the effects of PNT and cPNT on macrophages are mediated by NAL-insensitive receptors common for PNT, cPNT, IMN, and beta-END.

Asunto(s)

Activación de Macrófagos , Macrófagos/efectos de los fármacos , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacología , Receptores Opioides/agonistas , Secuencia de Aminoácidos , Animales , Unión Competitiva , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Regiones Constantes de Inmunoglobulina , Cadenas gamma de Inmunoglobulina , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Oligopéptidos/química , Oligopéptidos/metabolismo , Fragmentos de Péptidos/metabolismo , Péptidos/química , Péptidos/farmacología , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Fagocitosis , Receptores Opioides/metabolismo , betaendorfina/metabolismo

RESUMEN

Pulmonary inflammation is one of the risk factors associated with blood and marrow transplantation (BMT). To determine the potential role of T cells in pulmonary complications after transplantation, we analyzed the T-cell repertoire expressed in bronchoalveolar lavage fluids from eleven patients with graft-versus-host disease following BMT. A reverse transcriptase-polymerase chain reaction was used to amplify rearranged TCR transcripts in unfractionated, CD4+, and CD8+ T cells from bronchoalveolar lavage fluids. The relative expression of TCR variable (V) gene families and the diversity of junctional region lengths associated with different AV and BV gene families were analyzed. Nearly all TCR AV and BV gene families were detected in bronchoalveolar lavage cells from BMT recipients. Oligoclonal patterns of TCR junctional region lengths were observed in unfractionated, CD4+, and CD8+ bronchoalveolar T cells. The oligoclonal expansion of bronchoalveolar T cells in patients was confirmed by DNA sequencing. TCRV gene expression is almost completely restored in the lungs of BMT recipients as early as two weeks after transplantation. Increased oligoclonality among TCR gene families suggests either an incomplete restoration of TCR diversity or an antigen-driven expansion of T cells in the lungs of BMT recipients with graft-versus-host disease, not necessarily related to pulmonary infection.

Asunto(s)

Trasplante de Médula Ósea/inmunología , Enfermedad Injerto contra Huésped/inmunología , Enfermedades Pulmonares/inmunología , Pulmón/inmunología , Subgrupos de Linfocitos T/patología , Adulto , Trasplante de Médula Ósea/efectos adversos , Trasplante de Médula Ósea/patología , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Células Clonales/inmunología , Células Clonales/patología , Femenino , Reordenamiento Génico de Linfocito T , Supervivencia de Injerto , Enfermedad Injerto contra Huésped/patología , Humanos , Pulmón/patología , Enfermedades Pulmonares/etiología , Enfermedades Pulmonares/patología , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Trasplante Autólogo/efectos adversos , Trasplante Autólogo/inmunología , Trasplante Autólogo/patología , Trasplante Homólogo/efectos adversos , Trasplante Homólogo/inmunología , Trasplante Homólogo/patología

RESUMEN

OBJECTIVE: To examine domain recognition by anti-DNA topoisomerase I (anti-DNA topo I, or anti-topo I) antibodies over time in scleroderma patients. METHODS: Serial serum samples from scleroderma patients with known reactivity to Scl-70, a 70-kd topo I breakdown product, were tested by immunoblot for IgM, IgG, IgA, kappa, and lambda reactivity to Scl-70 and 8 overlapping recombinant peptide fragments (F1-F8) that span the human topo I molecule. RESULTS: IgM, IgG, kappa, and lambda anti-topo I antibodies in both early-disease and late-disease serum samples preferentially recognized the Scl-70 molecule rather than the F1-F8 peptides, suggesting preferential recognition of conformational determinants on Scl-70 throughout the disease course. Amounts of both primary and secondary anti-topo I antibodies to Scl-70 varied over time, including increases in primary antibody responses late in the disease course. Striking variability in recognition of the F1-F8 peptides by IgM, IgG, IgA, kappa, and lambda anti-topo I antibodies was seen in serial samples. Most often, the change in FI-F8 recognition from one sample to the next was unpredictable, although occasionally patterns of antibody recognition were reciprocal in serial samples. Of note, in several patients, what could have been interpreted as domain spreading among F1-F8 in 2 successive samples was just a part of changing antibody reactivity to these peptides that again became more restricted in a third sample. CONCLUSION: Titers and immunodominant domains recognized by both primary and secondary anti-topo I antibodies are highly variable over time. This suggests continual antigen presentation and regulation of the anti-topo I antibody response in scleroderma, even late in the disease course.

Asunto(s)

ADN-Topoisomerasas de Tipo I/inmunología , Esclerodermia Sistémica/inmunología , Adolescente , Adulto , Diversidad de Anticuerpos , Formación de Anticuerpos , Autoanticuerpos/inmunología , Femenino , Humanos , Sistema Inmunológico/metabolismo , Masculino , Persona de Mediana Edad , Esclerodermia Sistémica/sangre , Factores de Tiempo

RESUMEN

NPC 15669, a member of the leumedins family, inhibits leukocyte adhesion to the endothelium by blockage of upregulation of a member of beta2 integrin family Mac-1 (CD11b/CD18). Inhibition of neutrophil-endothelial interactions may alter the course of myocardial reperfusion injury. However, the effects of NPC 15669 supplementation on the hemostatic profile during ischemia-reperfusion are unknown. The aim of the present study was to define changes in the certain hemostatic factors in the natural course of preconditioned myocardial infarction. Twelve consecutive Yorkshire swine underwent myocardial stunning (8 min. left anterior descending artery occlusion followed by 90 min. of reperfusion) and then preconditioned myocardial infarction (50 min. occlusion followed by 3 hours of reperfusion) experiments. NPC 15669 (10 mg/kg loading dose followed by constant infusion at 6 mg x kg(-1) x h(-1)) was administered in 6 animals; another 6 swine received saline and served as controls. Blood samples were obtained at baseline, twice during occlusion; and three times during reperfusion. The levels of antithrombin-III, Protein C, total Protein S, fibronectin, endothelin-1, as well as the stable metabolites of thromboxane (TxB2) and prostacyclin (6-keto-PGF1a), were determined. NPC 15669 treatment was associated with diminished endothelin-1, TxB2 levels and increased fibronectin, 6-keto-PGF1a, Protein C and total Protein S concentrations in the setting of preconditioned myocardial infarction. There were no changes in the plasma concentrations of antithrombin-III in NPC 15669 group when compared with controls. The increase in Protein C, total Protein S, and 6-keto-PGF1a (favoring antithrombosis), and decrease in endothelin-1 and TxB2 levels (favoring vasodilatation), following NPC 15669 may explain the reduction in infarct size previously reported with this agent.

Asunto(s)

Hemostasis/efectos de los fármacos , Precondicionamiento Isquémico Miocárdico , Leucina/análogos & derivados , Antígeno de Macrófago-1/efectos de los fármacos , Infarto del Miocardio/tratamiento farmacológico , Animales , Antitrombina III/metabolismo , Evaluación Preclínica de Medicamentos , Epoprostenol/sangre , Femenino , Fibronectinas/sangre , Leucina/farmacología , Porcinos , Tromboxano B2/sangre

RESUMEN

OBJECTIVE: This study addresses the hypothesis that a profibrotic pattern of cytokines is produced in the lungs of patients with systemic sclerosis (SSc) and causes fibrosis. METHODS: Using a reverse transcriptase-polymerase chain reaction technique, interleukin-4 (IL-4), IL-5, and interferon-gamma (IFNgamma) messenger RNA (mRNA) were measured in unseparated CD8+ and CD4+ bronchoalveolar lavage (BAL) cells from SSc patients and healthy controls. To confirm the results, CD8+ T cells were cloned from BAL fluids, and the pattern of cytokine mRNA made by these cells was determined. Serial pulmonary function tests were done. RESULTS: BAL cells from healthy controls made IFNgamma mRNA, with no or little IL-4 or IL-5 mRNA. In contrast, BAL cells from the majority of SSc patients made IL-4 and/or IL-5 mRNA, with or without approximately equal amounts of IFNgamma mRNA. This pattern of cytokines was made by CD8+ T cells, which were increased in the lungs of these SSc patients. Patients whose BAL cells made this type 2 pattern of cytokine mRNA had a significant decline in forced vital capacity over time after the BAL, whereas patients whose BAL cells made IFNgamma mRNA alone did not. Both wild-type and an alternative splice variant of IL-4 mRNA were increased in BAL cells from SSc patients. Both forms of IL-4 stimulated alpha2(I) collagen mRNA in human dermal and lung fibroblasts. CONCLUSION: The type 2 pattern of cytokine mRNA produced by BAL cells from SSc patients differs from unopposed IFNgamma production found in healthy BAL cells. This production of type 2 cytokine mRNA by CD8+ T cells is associated with a significant decline in lung function over time, which suggests a pathologic role for these T cells in interstitial fibrosis in SSc.

Asunto(s)

Linfocitos T CD8-positivos/metabolismo , Interferón gamma/biosíntesis , Interleucina-4/biosíntesis , Interleucina-5/biosíntesis , Fibrosis Pulmonar/metabolismo , Esclerodermia Sistémica/metabolismo , Adolescente , Adulto , Anciano , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Recuento de Células , Cartilla de ADN/química , Femenino , Citometría de Flujo , Humanos , Interferón gamma/genética , Interleucina-4/genética , Interleucina-5/genética , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Fibrosis Pulmonar/fisiopatología , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esclerodermia Sistémica/fisiopatología

RESUMEN

Myocardial stunning is characterized by transient contractile dysfunction occurring subsequent to an episode of ischemia followed by reperfusion. Platelet activation and hemostatic abnormalities have been described in patients with unstable angina and acute myocardial infarction, however, their role in the pathogenesis of myocardial stunning is unknown. The purpose of this study was to determine if platelet aggregation and certain hemostatic factors change during myocardial stunning following brief coronary arterial occlusion. Nine Yorkshire swine underwent left anterior descending coronary artery occlusion for 8 minutes followed by 90 minutes of reperfusion. Blood samples were obtained at baseline, at 4 and 8 minutes of occlusion, and at 60 and 90 minutes of reperfusion. Platelet aggregability and concentrations of antithrombin III, protein C, protein S, fibronectin, endothelin 1, and the stable metabolites of thromboxane (TxB2) and prostacyclin (6-keto-PGF1a) were measured in systemic circulation. The occlusion phase was associated with a decline of endothelin 1 (-13.6%), and TxB2 (-19.6%), and elevation of antithrombin III (+40.2%) and protein C (+22.9%). Mild myocardial stunning was associated with a significant increase in platelet aggregation (+33.7%), endothelin-1 (+24.7%), 6-keto-PGF1a (+41.5%), TxB2 (+11.9%), and protein C (+42.3%) during the reperfusion phase. There were no changes in plasma fibronectin and total protein S. Thus, mild myocardial stunning following brief coronary artery occlusion is associated with substantial dynamic changes in platelet aggregability and certain hemostatic factors. These results may be relevant to understanding the mechanisms determining myocardial stunning and coronary arterial patency following reperfusion.

Asunto(s)

Hemostasis , Aturdimiento Miocárdico/sangre , Agregación Plaquetaria , Animales , Modelos Animales de Enfermedad , Femenino , Porcinos

RESUMEN

T cells play a pivotal role in initiating and orchestrating allergic responses in asthma. The goal of this work was to learn whether ragweed challenge in the lungs alters the T-cell repertoire expressed in the blood and lungs of atopic asthmatics. Analyses of cell numbers, differentials, and T-cell subsets in bronchoalveolar lavage (BAL) fluids showed that ragweed challenge was associated with preferential recruitment of CD4+ T cells into the lungs. A reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify T-cell receptor (TCR) gene transcripts from unfractionated, CD4+, and CD8+ T cells in blood and BAL fluids. As judged by RT-PCR, the usage of TCR Valpha and Vbeta gene families in BAL fluids was similar to that in blood. Ragweed challenge did not change the levels of expression of these V gene families. The clonality of T cells was estimated by analyzing the diversity of TCR V-(D)-J junctional region nucleotide lengths associated with each Valpha and Vbeta gene family, using sequencing gel electrophoresis. Most V gene families in blood and BAL fluids were associated with multiple junctional region lengths before and after ragweed challenge, indicating polyclonal expression. Some V gene families were expressed in an oligoclonal manner in unfractionated, CD4+, and CD8+ T cells in BAL fluids before ragweed challenge, as indicated by a few predominant junctional region lengths. The majority of these V gene families became polyclonal after challenge, compatible with polyclonal T-cell influx during inflammation immediately after ragweed challenge. However, some V gene families became oligoclonal or developed a new oligoclonal pattern of junctional region lengths in BAL T cells after ragweed challenge. Surprisingly, this occurred in both CD4+ and CD8+ T cells. In one of these instances, DNA sequencing of Vbeta21 junctional regions in CD8+ T cells confirmed a change from polyclonal to oligoclonal expression after ragweed challenge. These findings show that ragweed challenge is associated with polyclonal influx and oligoclonal activation of both CD4+ and CD8+ T cells in the lungs.

Asunto(s)

Asma/inmunología , Hipersensibilidad Inmediata/inmunología , Pulmón/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Linfocitos T/inmunología , Adulto , Alérgenos/inmunología , Animales , Asma/sangre , Líquido del Lavado Bronquioalveolar/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Gatos , Femenino , Humanos , Hipersensibilidad Inmediata/sangre , Masculino , Ácaros/inmunología , Polen/inmunología , Análisis de Secuencia de ADN

RESUMEN

The molecular model of IL-4 delta 2, a naturally occurring splice variant of human IL-4 with exons 1, 3, and 4 in an open reading frame, is described. The second exon codes the main part of the long loop AB connected the helices A and B in parallel superposition. Therefore the hydrophobic core and the native fold of the rest part of IL-4 delta 2 molecule could be preserved without any significant changes only in the case of revolution of the helix A relative to other helices. In the result, the dominated a left-handed four-helix bundle structure of IL-4 with an up-up-down-down structural pattern is transformed to the IL-4 delta 2 structure with a down-up-down-down structural pattern.

Asunto(s)

Interleucina-4/química , Modelos Moleculares , Humanos , Estructura Terciaria de Proteína , Relación Estructura-Actividad

RESUMEN

Our previous work showed that alternative splicing is used to make an inhibitory variant of human interleukin (IL)-4. Because of homology between IL-4 and IL-2 proteins and receptors, we tested whether alternative splicing is used to generate similar inhibitory variants of human IL-2. Messenger RNA from peripheral blood mononuclear cells was subjected to reverse transcription-polymerase chain reaction using IL-2 exon 1- and exon 4-specific primers. Two amplification products, named IL-2delta2 and IL-2delta3, were found in addition to the native IL-2 product. The IL-2delta2 cDNA sequence was identical to IL-2 cDNA throughout the entire coding region, except exon 2 was omitted by alternative splicing. In IL-2delta3 cDNA, the third exon of IL-2 was omitted by alternative splicing. Unlike IL-2, IL-2delta2 and IL-2delta3 did not stimulate T cell proliferation. However, both inhibited IL-2 costimulation of T cell proliferation, and both inhibited cellular binding of rhIL-2 to high affinity IL-2 receptors. Thus, IL-2 is the second cytokine that uses alternative splicing to generate variants that are competitive inhibitors.

Asunto(s)

Empalme Alternativo , Interleucina-2/genética , Interleucina-2/metabolismo , Receptores de Interleucina-2/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Variación Genética , Humanos , Interleucina-2/farmacología , Activación de Linfocitos/efectos de los fármacos , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Análisis de Secuencia de ADN , Linfocitos T/efectos de los fármacos

RESUMEN

The repertoire of variable alpha (AV) and beta (BV) TCR genes was compared in the peripheral blood and BAL fluid of five healthy individuals. Rearranged TCR transcripts were amplified by a reverse transcription-polymerase chain reaction, using oligonucleotide primers specific for 22 AV and 24 BV gene families. Nearly all AV and BV gene families were expressed in BAL T cells at levels similar to those in blood T cells. The diversity of AV and BV gene repertoire was examined further, testing the distribution of nucleotide lengths of TCR junctional regions. Most V gene families had a normal distribution of junctional region lengths in both blood and BAL T cells. Some gene families, particularly AV21 and BV9 in BAL samples, had a skewed banding pattern, with fewer bands or predominance of several bands. The limited diversity in TCR junctional region lengths was more prominent in CD8+ T cells from BAL fluids than from blood. CD4+ T cells also contributed to the limited diversity in BAL T cells. The oligoclonal expansion of bronchoalveolar CD8+ T cells was confirmed by sequence analysis of AV21-constant alpha (AC) and BV9-BC junctional regions in the blood and BAL cells. The levels of V gene expression and the diversity of junctional region lengths were very similar in T cells obtained from three separate lobes of one donor. In general, skewed patterns of TCR junctional region lengths were not consistent over time two donors, over periods of 3 and 17 months. Together, these data show that the T-cell repertoire is diverse within the lungs of normal humans, except for an oligoclonal predominance of a few V gene families in both CD4+ and CD8+ T cells. The T-cell repertoire in the lungs changes over time, which may reflect environmental exposures.

Asunto(s)

Líquido del Lavado Bronquioalveolar/inmunología , Familia de Multigenes/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/inmunología , Adolescente , Adulto , Secuencia de Bases , Recuento de Células Sanguíneas , Citometría de Flujo , Humanos , Masculino

RESUMEN

Cytokines produced in abnormal amounts or patterns contribute to many immunologically mediated human diseases. We describe a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay to measure interleukin (IL)1-2, IL-4, and interferon-gamma (IFN-gamma) mRNAs within the sample. Internal standard cRNAs and native cytokine mRNAs are reverse transcribed and then amplified by PCR in the same reaction tubes to control for tube-to-tube variability in these reactions. In contrast to systems that use a single multigene internal standard cRNA, this method uses separate internal standard cRNAs for IL-2, IL-4, and IFN-gamma, allowing independent dosing of the internal standards, which reduces the number of tubes processed and the amount of starting mRNA required. Internal standards are produced from cytokine cDNAs by the insertion of short segments of DNA. The same oligonucleotide primers are used to amplify internal standard and native cytokine cDNAs. Each internal standard cDNA and its matching native cytokine cDNA are amplified with equal efficiency. The RT-PCR products of the internal standards and native cytokines are distinguished by size. This technique can detect a twofold difference in mRNA levels. Examples of using this technique to measure cytokine mRNAs in peripheral blood mononuclear cells and in bronchoalveolar lavage cells are given.

Asunto(s)

Citocinas/análisis , Líquido del Lavado Bronquioalveolar/citología , Citocinas/biosíntesis , Citocinas/genética , Amplificación de Genes , Interferón gamma/genética , Interleucina-2/genética , Interleucina-4/genética , Reacción en Cadena de la Polimerasa/métodos , ARN Complementario , ARN Mensajero/análisis

RESUMEN

Pulmonary parenchymal involvement in SSc is characterized by alveolitis and interstitial fibrosis, with an increased number of CD8+ T cells in BAL fluids. This study analyzed the diversity of the alphabeta T-cell repertoire in peripheral blood and BAL fluids from seven SSc patients, looking for evidence of antigen-driven selection of T cells in the lungs. A reverse transcriptase-polymerase chain reaction technique was used to amplify rearranged TCR transcripts from unfractionated, CD4+, and CD8+ T cells. Nearly all AV and BV gene families were expressed in SSc patients and most had similar levels of expression in blood and BAL samples. Next, the diversity of TCR junctional region lengths was assessed, using sequencing gel electrophoresis. Many V gene families had a Gaussian distribution of their junctional region lengths. However, some V gene families had an abnormal pattern of junctional lengths, with skewing away from a Gaussian distribution, including predominance of one or two lengths. This suggests selected expansion of T cells expressing those V genes. Alterations in TCR junctional region lengths were most prominent in bronchoalveolar CD8+ T cells, with similar patterns of skewing in several patients and in one patient over time. Sequence analysis of AV14 and BV17 junctional regions confirmed the oligoclonal character of expansion of bronchoalveolar CD8+ T cells.

Asunto(s)

Linfocitos T CD8-positivos/clasificación , Linfocitos T CD8-positivos/inmunología , Pulmón/inmunología , Esclerodermia Sistémica/inmunología , Subgrupos de Linfocitos T/clasificación , Subgrupos de Linfocitos T/inmunología , Adulto , Secuencia de Bases , Líquido del Lavado Bronquioalveolar/citología , Células Clonales , Clonación Molecular , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Esclerodermia Sistémica/sangre , Transcripción Genética/genética

RESUMEN

A second species of interleukin-4 (IL-4) mRNA was identified using both a reverse transcription-polymerase chain reaction and an RNase protection assay. This novel IL-4 mRNA was 48 base pairs smaller than IL-4 mRNA, which is the size of IL-4 exon 2. Sequence data of cloned cDNA demonstrated that this variant contained IL-4 exons 1,3 and 4, with exon 1 spliced directly to exon 3 in an open reading frame. The entire protein encoding region of this variant, named IL-4 delta 2, was identical to IL-4 except for the omission of exon 2. IL-4 delta 2 mRNA was detected in all human peripheral blood mononuclear cells tested and in purified CD3+ T cells. Amounts of both IL-4 and IL-4 delta 2 mRNAs increased upon T cell activation, although IL-4 mRNA increased to a greater extent than IL-4 delta 2 mRNA did. Human IL-3, IL-5, IL-13, and granulocyte macrophage-colony stimulating factor did not use alternative splicing to delete exon 2. We speculate that IL-4 delta 2 may regulate IL-4 function.

Asunto(s)

Empalme Alternativo , Interleucina-4/genética , Secuencia de Aminoácidos , Secuencia de Bases , Exones , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Humanos , Interleucina-3/genética , Interleucina-5/genética , Datos de Secuencia Molecular , ARN Mensajero/análisis , Ribonucleasas/farmacología , Linfocitos T/metabolismo

RESUMEN

Alternative splicing of mRNA can generate protein isoforms that are preferentially expressed in different tissues or during different states of cell differentiation or activation. Protein isoforms may have different functions. In this study, we cloned, expressed, and tested functional effects of a naturally occurring splice variant of human IL-4, called IL-4 delta 2. In IL-4 delta 2, the second exon of IL-4 is omitted by alternative splicing, with exons 1, 3, and 4 joined in an open reading frame. We found that IL-4 delta 2 RNA is expressed in the PBMC of all donors tested, usually in lower amounts than IL-4 RNA. In contrast, IL-4 delta 2 RNA is expressed in much higher levels than IL-4 RNA in thymocytes and bronchoalveolar lavage cells, suggesting tissue specificity of expression. IL-4 delta 2 cDNA was expressed in yeast. Recombinant human (rh) IL-4 delta 2 was partially purified and found to be glycosylated, with a protein core of 13 to 15 kDa. Unlike rhIL-4, rhIL-4 delta 2 did not act as a costimulator for T cell proliferation. However, rhIL-4 delta 2 inhibited the ability of rhIL-4 to act as a T cell costimulator. Inhibition was independent of glycosylation and was not mediated by toxicity. Iodinated IL-4 delta 2 was found to bind specifically to human PBMC and tumor lines known to express IL-4 receptors. Excess unlabeled IL-4 inhibited cellular binding of labeled IL-4 delta 2. Thus, rhIL-4 delta 2 is a naturally occurring splice variant of IL-4 that is preferentially expressed in the thymus and airways and inhibits function of complete IL-4. The balance between IL-4 and IL-4 delta 2 may be important in the regulation of IL-4 effects.

Asunto(s)

Interleucina-4/antagonistas & inhibidores , Interleucina-4/genética , Activación de Linfocitos/efectos de los fármacos , Empalme del ARN , Linfocitos T/efectos de los fármacos , Secuencia de Aminoácidos , Antígenos CD/efectos de los fármacos , Antígenos CD/metabolismo , Secuencia de Bases , Líquido del Lavado Bronquioalveolar/citología , Células Cultivadas , Clonación Molecular , ADN Complementario/genética , Depresión Química , Exones/genética , Humanos , Interleucina-4/aislamiento & purificación , Interleucina-4/farmacología , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Especificidad de Órganos , ARN Mensajero/genética , Receptores de Interleucina/efectos de los fármacos , Receptores de Interleucina/metabolismo , Receptores de Interleucina-4 , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , Sistema Respiratorio/metabolismo , Timo/metabolismo

RESUMEN

Systemic sclerosis (SSc) is a multisystem disease characterized by T-cell infiltration of involved tissues, fibrosis, and small vessel vasculopathy. Using flow cytometric analyses, we found an increased percentage of gamma/delta T-cells expressing the T-cell antigen receptor variable (V) delta 1 gene segment in the peripheral blood and bronchoalveolar lavage fluid of patients with SSc. To estimate clonality of these V delta 1+ T-cells, the diversity of V delta 1 junctional regions (V-Diversity-Joining gene segments) was examined using a reverse transcriptase-polymerase chain reaction to amplify T-cell antigen receptor delta chain transcripts isolated from peripheral blood mononuclear cells, lung, esophagus, stomach, or skin of patients and controls. Limited diversity of V delta 1-J delta junctional regions in SSc patients was demonstrated by the finding of greater restriction in the nucleotide lengths of junctional region cDNAs in individual SSc patients than in controls. Sequence analyses confirmed that V delta 1-J delta junctional regions from the blood of SSc patients had less diversity than those from controls, in that a significantly higher proportion of sequences were repeated in patients (54.4% vs. 19.4% in controls). Evidence for selection of the V delta 1+ T-cells in tissues of individual SSc patients came from the findings that the same V delta 1-J delta junctional sequences could be isolated from the same tissue over time and that identical V delta 1-J delta junctional sequences could be isolated from multiple tissues. These data suggest that expansion of V delta 1+ gamma/delta T cells may be antigen driven in SSc patients.

Asunto(s)

Reordenamiento Génico de la Cadena delta de los Receptores de Antígenos de los Linfocitos T , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Esclerodermia Sistémica/inmunología , Linfocitos T/inmunología , Secuencia de Bases , Células Clonales , Cartilla de ADN/química , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Esclerodermia Sistémica/patología , Linfocitos T/patología

RESUMEN

An increase of certain T cell subsets in systemic sclerosis patients, particularly of V delta 1+ gamma delta T cells in the blood and lungs and CD8+ alpha beta T cells in the lungs, has been shown. The diversity of T cell antigen receptor (TCR) V delta 1, V alpha, and V beta gene repertoires was examined using reverse transcriptase-polymerase chain reaction to amplify rearranged TCR transcripts across the junctional region. This was followed by two methods of analysis. First, the relative expression of V alpha and V beta genes was determined in the blood and bronchoalveolar lavage fluid of the patients. Second, we looked for evidence of restricted diversity of the junctional regions in TCR V delta 1 transcripts and in different V alpha and V beta gene families. Limited V delta 1-C delta junctional region lengths were observed in the patients compared to controls. This was confirmed by sequence analysis of V delta 1-C delta junctional regions after subcloning amplified products in a bacterial vector. A restricted diversity of the junctional region lengths was also detected in a number of V alpha and V beta gene families, particularly within bronchoalveolar CD8+ T cell subset. These data suggest that the oligoclonal expansion of the corresponding alpha beta and gamma delta T cells is antigen-driven and may be important in the pathogenesis of systemic sclerosis.

Asunto(s)

Esclerodermia Sistémica/genética , Esclerodermia Sistémica/inmunología , Subgrupos de Linfocitos T/inmunología , Sangre/inmunología , Humanos , Pulmón/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología

RESUMEN

Certain T cell subsets are increased in systemic sclerosis patients, particularly V delta 1+ gamma delta T cells in the blood and lungs and CD8+ alpha beta T cells in the lungs. The repertoires of T cell antigen receptor (TCR) V delta 1, V alpha, and V beta gene families were examined by two methods of analysis. First, the relative abundance of V alpha and V beta gene transcripts was determined in the blood and bronchoalveolar fluid of the patients. Second, the diversity of the junctional regions in TCR V delta 1 transcripts and in different V alpha and V beta gene families was analyzed. Limited V delta 1-C delta junctional region lengths were observed in the patients compared with controls. This was confirmed by sequence analysis of V delta 1-C delta junctional regions after subcloning amplified products in a bacterial vector. Evidence for selection of the V delta 1+ T cells in the tissues of patients came from the findings that the same V delta 1-C delta junctional sequences persisted in an individual patient over time and that identical junctional sequences were isolated from multiple sites. A restricted diversity of the junctional region lengths was also detected in a number of V alpha and V beta gene families, particularly within bronchoalveolar CD8+ T cell subset. These data suggest that the oligoclonal expansion of the corresponding alpha beta and gamma delta T cells is antigen-driven and may be important in the pathogenesis of systemic sclerosis.

Asunto(s)

Receptores de Antígenos de Linfocitos T/clasificación , Esclerodermia Sistémica , Humanos