RESUMEN

The anti-inflammatory properties of a novel pyrrolopyrimidine, PNU-142731A, in a murine model of antigen-induced eosinophilic lung inflammation are described. PNU-142731A, when given orally, demonstrated a dose-related inhibition of eosinophil- and lymphocyte-rich accumulation in the airways of ovalbumin (OA)-sensitized and challenged (OA/OA) C57BL/6 mice. The magnitude of the suppression of lung inflammation was also dependent on length of treatment. Reductions in the levels of interleukin (IL)-5, IL-6, and IgA in the bronchoalveolar lavage fluid of treated OA/OA mice, when compared with vehicle-sensitized control mice (V/OA), were observed. Plasma concentrations of IL-5, total IgE, and OA-specific IgG1 were also lowered in OA/OA mice by treatment. Histological assessment of formalin-fixed lung tissue sections confirmed that the compound blocked the accumulation of eosinophils in the airway tissue. Furthermore, significantly less mucus glycoproteins were seen in the lungs of PNU-142731A-treated OA/OA mice. Reverse transcription-polymerase chain reaction of lung tissue from PNU-142731A-dosed OA/OA mice demonstrated that mRNA for Th2 cytokines was less than that in vehicle-treated OA/OA controls. OA-elicited production of IL-4 by disaggregated lung tissue cells from PNU-142371A-treated OA/OA mice was also less than that of controls. In contrast, the release of Th1 cytokines (IL-2 and interferon-gamma) were elevated. Similarly, the OA-stimulated release of Th2 cytokines (IL-5 and IL-10) by splenocytes from PNU-142731A-treated OA/OA mice were inhibited. Combined therapy of OA/OA mice with PNU-142731A and suboptimal doses of dexamethasone revealed that PNU-142731A had steroid-sparing effects. These characteristics of PNU-142731A in a murine model of allergic tissue inflammation support its clinical development as a potential treatment for asthma.

Asunto(s)

Antiasmáticos/farmacología , Antiinflamatorios no Esteroideos/farmacología , Indoles/farmacología , Pirrolidinas/farmacología , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/biosíntesis , Citocinas/genética , Citocinas/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Inmunoglobulina A/metabolismo , Inmunoglobulina E/metabolismo , Inmunoglobulina G/metabolismo , Inmunoglobulinas/biosíntesis , Técnicas In Vitro , Leucocitos/efectos de los fármacos , Leucocitos/inmunología , Leucocitos/metabolismo , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Moco/metabolismo , Ovalbúmina/inmunología , ARN Mensajero/biosíntesis , Sistema Respiratorio/efectos de los fármacos , Sistema Respiratorio/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidores de Serina Proteinasa/farmacología , Bazo/citología , Bazo/efectos de los fármacos , Bazo/metabolismo

RESUMEN

We investigated the effects of in vivo intraperitoneal treatment with the rat monoclonal antibody (mAb), YN1.7.4 (YN1) against intercellular adhesion molecule-1 (ICAM-1) on the ovalbumin (OA)-inhalation-induced infiltration of leukocytes into the airways of OA-sensitized mice. YN1 (100 to 400 microg) given over a period of 72 h dose-dependently reduced the influx of lymphocytes and eosinophils into the bronchial lumen by > 60% and > or = 70%, respectively, when compared with saline or purified rat IgG-treated controls. Alveolar macrophages (AM) in the bronchoalveolar lavage fluid (BALF) were also decreased by > 50%. Lung tissue inflammation as determined by histopathologic examination was reduced. The number of neutrophils in the blood of OA-sensitized mice 3 days after challenge was significantly increased by treatment with YN1. However, at 24 h and 72 h after OA-challenge, the numbers of eosinophils and mononuclear cells in the bone marrow were reduced by YN1 treatment. Additionally, at 72 h after OA-challenge, the numbers of bone-marrow neutrophils were depressed. BALF levels of interleukin-5 (IL-5) and of IgA were lower for YN1-treated mice than for controls. With increasing doses of YN1, the levels of anti-ICAM-1 mAb in the plasma were proportionally increased. To correlate these results with YN1 treatment, blood and BALF T cells and BALF eosinophils were examined with flow cytometry. Blood T cells from YN1-treated mice were unable to bind phycoerythrin (PE)-labeled anti-ICAM- mAb ex vivo. These results implied that ICAM-1 on these cells was bound (occupied) by YN1 administered in vivo. Dose-related decreases were observed in the percentage and mean channel fluorescence (MCF) values of ICAM-1+ BALF T cells and eosinophils. The percentages of CD11a+ or CD49d+ eosinophils were also suppressed. Our data suggest that ICAM-1 is an important molecule involved in the recruitment of leukocytes into the airways of sensitized mice after pulmonary challenge.

Asunto(s)

Líquido del Lavado Bronquioalveolar/inmunología , Eosinófilos/inmunología , Molécula 1 de Adhesión Intercelular/inmunología , Pulmón/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/metabolismo , Antígenos/inmunología , Células de la Médula Ósea , Quimiotaxis de Leucocito/inmunología , Modelos Animales de Enfermedad , Eosinófilos/metabolismo , Femenino , Inmunoglobulina A/análisis , Interleucina-5/análisis , Leucocitos/inmunología , Macrófagos Alveolares/inmunología , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/inmunología , Eosinofilia Pulmonar/inmunología , Ratas , Linfocitos T/metabolismo

RESUMEN

The role of intercellular adhesion molecule-1 (ICAM-1) in murine lung inflammation was examined in vivo. Ovalbumin (Ova)-sensitized and -challenged ICAM-1-deficient (KO) mice had decreased accumulation of leukocytes in the bronchoalveolar lavage fluid compared with wild-type (WT) mice. Lung tissue inflammation was also attenuated. Ova immunization and challenge produced equivalent plasma levels of Ova-specific immunoglobulin (Ig) G1 and higher concentrations of IgE in KO versus WT mice. Ova-dependent induction of cytokines in vitro, as measured by enzyme-linked immunosorbent assay, was impaired in splenocytes from KO mice compared with the comparable release of interleukin (IL)-5 and IL-10 from anti-CD3-stimulated WT and KO splenocytes. Methacholine-induced increases in trapped gas in lungs of Ova-sensitized and -challenged WT mice were greater than those of KO mice. The activation of lung tissue nuclear factor-kappa B was diminished in KO mice after Ova provocation. This suggests that ICAM-1 was important for activation of the inflammatory cascade leading to the recruitment of leukocytes but was not critical for the generation of antibody responses in vivo.

Asunto(s)

Citocinas/biosíntesis , Inmunoglobulina E/biosíntesis , Inmunoglobulina G/biosíntesis , Molécula 1 de Adhesión Intercelular/fisiología , Leucocitos/fisiología , Pulmón/fisiología , Linfocitos T/inmunología , Animales , Formación de Anticuerpos , Antígenos de Diferenciación/análisis , Líquido del Lavado Bronquioalveolar/citología , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Inflamación , Molécula 1 de Adhesión Intercelular/genética , Leucocitos/inmunología , Pulmón/inmunología , Pulmón/patología , Cloruro de Metacolina/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Ovalbúmina , Bazo/inmunología

RESUMEN

We have used a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect the expression of mRNA for inflammatory cytokines, integrins and selectins in murine lung tissue, and T cells and eosinophils isolated from lung and bronchoalveolar lavage (BAL) fluid in an in vivo model of ovalbumin (OA)-induced airway inflammation. RNA was isolated from whole lung tissue at 1, 6, 24, 48, 72 h, and 7 days after OA inhalation. mRNA for the Th2 cytokines, IL-4, -5, -6, -10 and -13 in OA-sensitized mice were significantly elevated compared with non-sensitized mice. IL-2, TNF-beta, and eotaxin mRNA were also increased, but IFN-gamma mRNA was not. P- and E-selectin mRNA levels were also enhanced in lung tissue between 6 and 72 h after challenge. Lung T cells were isolated by cell sorting with a flow cytometer at 3, 12, 24, 48 and 72 h after challenge. mRNA levels for IL-5 and -10 were greater in T cells from OA-sensitized and -challenged mice than controls at 24 h. BAL fluid from OA-sensitized and -challenged mice also had significantly higher IL-5 levels than controls. BAL fluid T cells and eosinophils were obtained at 48 and 72 h after aerosol challenge and were purified by cell sorting. Messenger RNA for IL-1 alpha, -2, -4, -5, -10, IFN-gamma, and beta 1 were detected in T cells at both time points. Transcripts for IL-1 alpha, -4, -5, -13, TNF-alpha and beta, and alpha 4, beta 1 and beta 7 were also identified in BAL eosinophils. These data show that in addition to murine lung T cells, airway eosinophils may also contribute to the inflammatory response by their ability to express mRNA for cytokines and integrins.

Asunto(s)

Moléculas de Adhesión Celular/genética , Quimiocinas CC , Citocinas/genética , Eosinófilos/química , Cadenas beta de Integrinas , Pulmón/citología , Linfocitos T/química , Animales , Antígenos CD/genética , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Separación Celular , Quimiocina CCL11 , Factores Quimiotácticos Eosinófilos/genética , Citocinas/metabolismo , Selectina E/genética , Eosinófilos/citología , Eosinófilos/inmunología , Femenino , Inflamación/metabolismo , Integrina alfa4 , Integrina beta1/genética , Integrinas/genética , Interferón gamma/genética , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-13/genética , Interleucina-13/metabolismo , Interleucina-2/genética , Interleucina-4/genética , Interleucina-4/metabolismo , Interleucina-5/genética , Interleucina-5/metabolismo , Linfotoxina-alfa/genética , Ratones , Ratones Endogámicos C57BL , Selectina-P/genética , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisis , Linfocitos T/citología , Linfocitos T/inmunología

RESUMEN

The involvement of the alpha4-integrin very late activation antigen 4 and vascular cell adhesion molecule-1 (VCAM-1) in leukocyte trafficking into the airways of ovalbumin (OA)-sensitized and OA-challenged mice was investigated using in vivo administration of anti-alpha4 monoclonal antibodies (mAb) PS/2, R1-2, and M/K-2.7 (MK2), specific for VCAM-1. VCAM-1 was upregulated on endothelial cells in lung tissue after OA inhalation. PS/2, R1-2, or MK2 significantly inhibited the recruitment of eosinophils and lymphocytes into the bronchoalveolar lavage (BAL) fluid and decreased inflammation in the lung tissues. Escalating in vivo doses of PS/2 or MK2 increased circulating levels of rat immunoglobulin G in the plasma. The binding of phycoerytherin-labeled anti-alpha4 mAb to blood T cells from PS/2-treated mice was reduced, implying that alpha4 sites were already occupied. T cells and eosinophils in BAL fluid from mice treated with PS/2 or MK2 were phenotypically different from controls. Selective decreases of alpha4+ T cells in the BAL fluid after PS/2 or MK2 treatment were coupled with changes in CD8+, CD11a, and CD62L expression. The alpha4-integrin and VCAM-1 may have important roles in the antigen-induced recruitment of T cells and eosinophils during OA-induced airway inflammation. The data suggest that these adhesion molecules may be suitable targets for therapeutic intervention in certain conditions of pulmonary inflammation.

Asunto(s)

Antígenos CD/fisiología , Leucocitos/fisiología , Pulmón/fisiopatología , Molécula 1 de Adhesión Celular Vascular/fisiología , Animales , Anticuerpos Monoclonales , Bronquios/patología , Movimiento Celular , Femenino , Inmunización , Inmunohistoquímica/métodos , Integrina alfa4 , Leucocitos/patología , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/inmunología , Neumonía/inmunología , Neumonía/patología , Ratas , Coloración y Etiquetado

RESUMEN

We used flow cytometry and treatment in vivo with a monoclonal antibody (mAb), TA-2, to the alpha 4 integrin to investigate the role of alpha 4 beta 1, CD49d/CD29 (VLA-4) in antigen-induced lung inflammation in Brown Norway (BN) rats. Ovalbumin (OVA) inhalation induced an accumulation of eosinophils and lymphocytes in the lungs and bronchoalveolar lavage (BAL) fluid of sensitized BN rats at 24 h after challenge. Phenotypic analyses demonstrated that the percentages of T cells expressing detectable alpha 4 and CD25 in the bronchial lumen after antigen challenge were dramatically increased compared with blood and lymph node T cells. The mean channel fluorescence values of alpha 4 expression were also increased on BAL T cells compared with blood or lymph node T cells. Treatment of OVA-sensitized rats in vivo with total cumulative doses of 0.75 to 6 mg/kg TA-2 mAb intraperitoneally produced dose-related increases in circulating TA-2 and a peripheral blood lymphocytosis, basophilia, and eosinophilia. Flow cytometric analysis of the peripheral blood T cells after in vivo TA-2 mAb administration showed decreases in detectable alpha 4 when these cells were examined ex vivo. Treatment with TA-2, but not an isotype-matched control mouse immunoglobulin G1 mAb, markedly inhibited the OVA-induced recruitment of lymphocytes and eosinophils into the airway lumen. Very few CD3+CD49d+ cells migrated into BAL fluid following anti-alpha 4 mAb treatment in vivo. Treatment with TA-2 also significantly attenuated OVA-induced inflammatory histopathology. We conclude that VLA-4 is a critically important adhesion molecule involved in antigen-specific lung inflammation in sensitized BN rats.

Asunto(s)

Antialérgicos/inmunología , Eosinófilos/inmunología , Integrinas/fisiología , Pulmón/inmunología , Receptores Mensajeros de Linfocitos/fisiología , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Líquido del Lavado Bronquioalveolar/citología , Eosinófilos/citología , Citometría de Flujo , Inmunofenotipificación , Integrina alfa4beta1 , Recuento de Leucocitos , Pulmón/citología , Subgrupos Linfocitarios/inmunología , Tejido Linfoide/citología , Masculino , Ratones , Ovalbúmina/inmunología , Neumonía/inmunología , Neumonía/patología , Ratas , Ratas Endogámicas BN , Hipersensibilidad Respiratoria/inmunología , Linfocitos T/citología

RESUMEN

We investigated the involvement of intercellular adhesion molecule-1 (ICAM-1; CD54) in ovalbumin (OA) antigen-induced lung inflammation in sensitized Brown Norway (BN) rats by using flow cytometry and in vivo treatment with a murine monoclonal antibody (MAb), 1A29, directed against rat ICAM-1. OA-challenge induced an eosinophil and lymphocyte-rich accumulation of leukocytes into the airway lumen. Between 75 and 90% of the T cells in bronchoalveolar lavage (BAL) fluid after challenge expressed CD54 and CD11a and were of the memory phenotype. 1A29 treatment produced dose-related increases in circulating 1A29 and blood neutrophils. In the BAL fluid of 1A29-treated animals, significant (P < 0.05) reductions in the numbers of eosinophils and lymphocytes, but not neutrophils or alveolar macrophages, were observed in association with a reduced inflammatory pathology in lung tissue. 1A29 administration reduced the number of detectable ICAM-1 binding sites on T cells in peripheral blood and BAL fluid examined ex vivo by flow cytometry. We conclude that ICAM-1 is critically important for the antigen-specific recruitment of eosinophils and lymphocytes into the lungs.

Asunto(s)

Antígenos/inmunología , Molécula 1 de Adhesión Intercelular/fisiología , Neumonía/inmunología , Animales , Anticuerpos Monoclonales , Células Sanguíneas/fisiología , Líquido del Lavado Bronquioalveolar/citología , Inmunización , Pulmón/inmunología , Pulmón/patología , Tejido Linfoide/patología , Ovalbúmina/inmunología , Fenotipo , Neumonía/patología , Ratas , Ratas Endogámicas BN , Linfocitos T/fisiología

RESUMEN

In order to investigate whether the pulmonary response to helminth antigens mimics that seen in allergic inflammation of the airways, we have examined the phenotypic characteristics of lymphocytes and eosinophils recruited to the airways following Nippostrongylus brasiliensis (N.b.) infection. Specifically, the cellular response was divided into an early and a late phase. During the early response there was a small but significant increase in neutrophil numbers recovered by bronchoalveolar lavage (BAL). Phenotypic analysis of BAL leukocytes revealed an early rise in the percentage of BAL lymphocytes expressing the naive T cell markers CD45RB and L-selectin, and the activation marker IL-2R. In addition, during the early response, there was an increased percentage of lymphocytes expressing the gamma delta TCR, but not the alpha beta TCR. In contrast, the late response was marked by a much larger accumulation, in the lungs and BAL, of memory CD4+ T lymphocytes and an influx of small, hypodense eosinophils which produced LTB4 and LTC4 on stimulation with calcium ionophore. At this time there was a substantial increase in the number of T lymphocytes and eosinophils expressing ICAM-1 and the integrins VLA-4 and LFA-1, implicating these adhesion molecules in inflammatory cell recruitment to the airways. We conclude that the pattern and phenotypic characteristics of the cellular recruitment seen following N.b. infection resemble those seen in early- and late-phase allergic inflammation of the airways in asthma, and therefore N.b. may be used to model these aspects of the disease.

Asunto(s)

Eosinófilos/patología , Neumonía/inmunología , Linfocitos T/patología , Animales , Líquido del Lavado Bronquioalveolar/citología , Moléculas de Adhesión Celular/análisis , Modelos Animales de Enfermedad , Eicosanoides/biosíntesis , Eicosanoides/inmunología , Eosinófilos/metabolismo , Eosinófilos/microbiología , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Inmunofenotipificación , Recuento de Leucocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Nippostrongylus/inmunología , Neumonía/microbiología , Neumonía/patología , Ratas , Ratas Sprague-Dawley , Infecciones por Strongylida/inmunología , Linfocitos T/química , Linfocitos T/microbiología , Factores de Tiempo

RESUMEN

Cytokines released from CD4+ T lymphocytes contribute to the pathogenesis of asthma by influencing the differentiation and function of eosinophils, the primary effector cells that cause airway epithelial damage. Using a model of ovalbumin (OA)-induced, eosinophil-rich chronic lung inflammation in sensitized mice, we have defined the role of T lymphocytes further by using three-color flow cytometry to characterize the adhesion and activation antigens that may be associated with the migration of these cells into the lung and airway lumen. OA inhalation in OA-sensitized C57BL/6 mice resulted in an early (6 to 24 h) influx of neutrophils into the bronchial lumen as enumerated by bronchoalveolar lavage (BAL), which was followed by a marked accumulation of lymphocytes and eosinophils between 24 to 72 h. Phenotypic analysis of BAL or lung tissue T cells showed that most Thy-1 CD3+ T cells were CD4+ (CD4: CD8 ratio of 3 to 4:1). The majority (90%) of the T cells in lung or BAL fluid expressed alpha beta T-cell receptors (TCR). Only 3 to 7% of the T cells were gamma delta TCR+ even though almost 25% of the T cells were CD4- CD8-. There were very few natural killer (NK) or B cells in BAL fluid compared with 15% B cells in dissagregated lung tissue. In contrast to T cells in spleen, almost all the lung and BAL T cells were of the memory phenotype, as ascertained by the expression of high levels of CD44 and by the absence of L-selectin and CD45RB on the cell surface. Fifty to ninety percent of lung and BAL T cells from vehicle-sensitized or OA-sensitized and challenged mice expressed the adhesion molecules CD11a (LFA-1), CD54 (ICAM-1), and CD49d (VLA-4). The early T-cell activation marker CD69 was upregulated on 30% of the lung and BAL T cells in OA-sensitized mice after antigen inhalation. When BAL fluid T cells from OA-sensitized and challenged mice were analyzed for their coexpression of adhesion and/or activation molecules, 75% of the cells that expressed one of three adhesion molecules, CD54, CD49d, or CD11a, also expressed at least one of the other two antigens. At least 15% of BAL T cells had all three of these molecules on their cell surfaces. The OA-dependent, temporally regulated emigration of T cells into the bronchial lumen after exposure to aerosolized antigen may be correlated with the accumulation of cells that express the memory phenotype with enhanced expression of adhesion molecules.

Asunto(s)

Pulmón/patología , Ovalbúmina/inmunología , Sistema Respiratorio/patología , Linfocitos T/inmunología , Administración Intranasal , Animales , Presentación de Antígeno , Moléculas de Adhesión Celular/análisis , Movimiento Celular , Femenino , Inmunofenotipificación , Inflamación/inmunología , Inflamación/patología , Pulmón/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/administración & dosificación , Sistema Respiratorio/inmunología , Linfocitos T/patología

RESUMEN

The IL-1R antagonist protein (IRAP) is a competitive inhibitor of IL-1, which is predominantly synthesized by monocytes. We show that this molecule is also expressed in human synovial fibroblasts and dermal fibroblasts (CRL 1445). IRAP mRNA was regulated in a time- and dose-dependent manner by IL-1 alpha, TNF-alpha, LPS, and PMA. Maximal induction of IRAP mRNA was observed between 8 and 16 h after stimulation with IL-1 alpha (1 U/ml), TNF-alpha (10 U/ml), LPS (50 ng/ml), and PMA (10 ng/ml). Their relative efficacy was as follows: PMA > LPS > IL-1 alpha > TNF-alpha. Potentiation was observed when fibroblasts were treated with IL-1 alpha plus basic fibroblast growth factor and IL-1 alpha plus platelet-derived growth factor-BB homodimer. Although LPS and PMA were the best inducers of IRAP mRNA, quantitation of the IRAP protein revealed that its synthesis and release were differentially regulated. Immunoprecipitation and SDS-PAGE of culture supernatant from LPS-treated cells and cell lysates of fibroblasts treated with LPS or PMA showed a single IRAP band with a molecular mass of approximately 22 kDa. Very little IRAP was detected in culture supernatants of cells treated with PMA. Quantitation of IRAP revealed that LPS induced the synthesis of secreted IRAP that was released, whereas the majority of the protein induced by PMA remained cell-associated. Reverse transcriptase-polymerase chain reaction amplification demonstrated that although LPS and PMA induced both transcripts, LPS preferentially induced secreted IRAP, whereas PMA differentially induced intracellular IRAP mRNA. Fibroblasts synthesize at least two different forms of IRAP depending on the inducing signal, and may regulate the inflammatory response by dampening the proinflammatory effects of IL-1 via a negative feedback mechanism with IRAP. The relative importance of fibroblast sIRAP vs intracellular IRAP in regulating the inflammatory response by the connective tissue remains to be determined.

Asunto(s)

Fibroblastos/metabolismo , Interleucina-1/antagonistas & inhibidores , Sialoglicoproteínas/genética , Secuencia de Bases , Células Cultivadas , Medios de Cultivo Condicionados , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica , Sustancias de Crecimiento/farmacología , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/farmacología , Lipopolisacáridos/farmacología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Sialoglicoproteínas/análisis , Piel/citología , Piel/metabolismo , Membrana Sinovial/citología , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

We studied the adhesion of human peripheral blood T lymphocytes to human synovial fibroblasts stimulated with interferon-gamma (IFN gamma), tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), or combinations of these cytokines. T lymphocytes bound poorly to untreated human synovial fibroblasts. IFN gamma treatment resulted in the largest increase in adhesion, followed by TNF alpha and IL-1 beta. Combinations of IFN gamma + TNF alpha and IFN gamma + IL-1 beta had a synergistic effect on intercellular adhesion molecule 1 (ICAM-1) expression and adhesion. The increase in cellular adhesion induced by cytokines correlated with the up-regulation of the number of cells expressing ICAM-1 and the density of antigen/cell. There was no synergistic effect on leukocyte function-associated antigen 3 (LFA-3) or on HLA class I or class II antigen expression. Adhesion was only partially inhibited by anti-ICAM-1, anti-LFA-1, or anti-CD18. These findings suggest the existence of ICAM-1--independent and CD11/CD18-independent adhesion mechanisms. Anti-LFA-3 was completely ineffective as an inhibitor of adhesion. There was no additive or synergistic advantage of using combinations of antibodies to increase the level of inhibition, i.e., anti--ICAM-1 + anti-LFA-3, anti-ICAM-1 + anti-CD18, or anti-ICAM-1 + anti-LFA-1 (CD11a). Our data indicate that proinflammatory cytokines may play a prominent role in the formation and exacerbation of synovial hyperplasia, by regulating the recruitment and retention of T lymphocytes via the up-regulation of adhesion molecules on synovial fibroblasts.

Asunto(s)

Antígenos CD/metabolismo , Adhesión Celular/inmunología , Citocinas/inmunología , Fibroblastos/inmunología , Receptores de Adhesión de Leucocito/inmunología , Membrana Sinovial/inmunología , Linfocitos T/inmunología , Anticuerpos/inmunología , Antígenos CD/inmunología , Antígenos CD18 , Moléculas de Adhesión Celular/inmunología , Células Cultivadas , Citocinas/farmacología , Citometría de Flujo , Humanos , Molécula 1 de Adhesión Intercelular , Interferón gamma/farmacología , Interleucina-1/farmacología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Sinovitis/inmunología , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba/inmunología

RESUMEN

The interactions of interleukin-1 beta (IL-1 beta) and basic fibroblast growth factor (bFGF) with monolayer cultures of rabbit articular chondrocytes were examined. Both agonists resulted in a synergistic increase in prostanoid production, to levels higher than the maximal level for either protein alone. The synergy was time- and dose-dependent, was augmented in serum-free medium, and was blocked by indomethacin or a polyclonal antibody to IL-1 beta. The proteins had opposite effects on cell growth, and IL-1 beta completely blocked the mitogenic effect of bFGF. Pretreatment of cells with IL-1 beta induced a down-regulation in the number of the bFGF high-affinity receptors. Pretreatment of cells with bFGF increased the number of IL-1 receptors, which was dependent on messenger RNA synthesis.

Asunto(s)

6-Cetoprostaglandina F1 alfa/biosíntesis , Cartílago Articular/citología , Dinoprostona/biosíntesis , Factor 2 de Crecimiento de Fibroblastos/farmacología , Interleucina-1/farmacología , Receptores de Superficie Celular/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Cartílago Articular/metabolismo , División Celular , Células Cultivadas , Sinergismo Farmacológico , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Interleucina-1/metabolismo , Masculino , Conejos , Receptores de Factores de Crecimiento de Fibroblastos , Receptores de Interleucina-1

RESUMEN

The effects of the in vivo administration of interleukin 1 (IL 1) on lymphocytes from lymph node and spleen were analyzed. Mice received five daily subcutaneous (s.c.) injections of various doses of human recombinant IL 1 beta. Either 1 or 7 days after IL 1 treatment, spleens, popliteal and inguinal lymph nodes were collected. Lymphadenosis and splenomegaly were observed in the IL 1-treated animals. Lymph nodes from IL 1-treated mice contained a higher percentage of B cells than controls, and B cells from IL 1-treated mice expressed dramatically increased levels of Ia antigen. Lymphadenosis and splenomegaly, as well as the changes in subset distributions and Ia expression were transient. Concomitant treatment of mice with IL 1 and anti-IL 4 monoclonal antibody suppressed IL 1 effects on B cell Ia expression, but not on the B/T cell ratio. In situ hybridization analyses revealed that IL 1 treatment induced the expression of mRNA for IL 4, interferon-gamma, and IL 2 in lymph node and spleen cells. The distribution of cells expressing the various cytokine mRNA was markedly different between the spleens and lymph nodes.

Asunto(s)

Linfocitos B/inmunología , Antígenos de Histocompatibilidad Clase II/análisis , Interleucina-1/farmacología , Interleucina-4/fisiología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Antígenos de Diferenciación/análisis , Linfocitos T CD4-Positivos/inmunología , Regulación de la Expresión Génica , Técnicas Inmunológicas , Interferón gamma/genética , Interleucina-2/genética , Interleucina-4/genética , Linfadenitis/inducido químicamente , Ratones , Ratones Endogámicos DBA , ARN Mensajero/genética , Esplenomegalia/inducido químicamente

RESUMEN

Immunofluorescence was used to characterize the lymphocyte subpopulations of mice treated with six immunomodulatory drugs: hydrocortisone acetate (HCA), corticosterone acetate (corticosterone), cyclophosphamide, cytosine arabinoside (Ara-C), 15(S)-methyl prostaglandin E1 (15(S)-methyl PGE1), and 2-amino-5-bromo-6-phenyl-4-(3H)-pyrimidinone (ABPP). The number of thymus and spleen cells bearing Thy-1, Ig, Lyt-1 and Lyt-2 antigens and the density of the antigens on each cell (IF profiles) were determined. Microscopic examination of cells stained with rhodamine-labeled anti-Lyt-2 and fluorescein-labeled anti-Lyt-1 was used to measure the proportion of Lyt-1+2-, Lyt-1+2+, and Lyt-1-2+ cells in the spleen and thymus of drug-treated animals. The changes in lymphocyte subpopulations were compared with the varied effects of these drugs on antibody formation and graft vs host (GVH) reaction. Three immunosuppressive drugs, HCA, cyclophosphamide, and Ara-C, depleted the thymus of cells expressing a large quantity of Thy-1. The drug-resistant cells were larger and had more Lyt-1 than cells from control animals. HCA treatment depleted the thymus of Lyt-1+2+ cells; the cortisone resistant cells were primarily Lyt-1+2-. Cyclophosphamide and the antiviral immunostimulant, ABPP, caused similar, but less marked, alterations. The proportion of Lyt-1-2+ cells in the thymus was reduced by treatment with all the drugs, but the density of Lyt-2 on the drug-resistant cells was not altered. Treatment with Ara-C or 15(S)-methyl PGE1 produced a very modest evaluation in Lyt-1+2- cells. 15(S)-Methyl PGE1, which suppresses some immuno-inflammatory reactions, had no discernible effect on thymocyte size or the IF profile of Thy-1, Lyt-1, or Lyt-2. In the spleen, the amount of Thy-1 and of immunoglobulin on cells bearing these markers was changed very little by drug treatment. The proportion of splenic B cells was diminished by treatment with cyclophosphamide and, to a lesser extent, by HCA, while the proportion of spleen cells bearing detectable Thy-1 and Lyt-1 increased correspondingly. The proportion of cells bearing Lyt-2 was altered by only two drugs; cyclophosphamide increased both Lyt-1+2+ and Lyt-1-2+ spleen cells and ABPP (an interferon inducer which stimulates antibody formation) decreased both Lyt-2+ subpopulations. Treatment with two drugs caused the serum corticosterone concentration to rise: ABPP increased serum corticosterone substantially while the prostaglandin induced a smaller and more transitory increase. An indirect mechanism, via corticosteroid release, might explain the thymic depletion observed in mice treated with 15(S)-methyl PGE1 and ABPP, but neither the suppression of the GVH reaction by these drugs nor polyclonal activation of B cells by ABPP can be attributed to endogenous corticosteroids. Our data show that enumeration of splenic lymphocyte subpopulations by immunofluorescence techniques may aid in elucidating the mode of action of immunomodulatory drugs.

Asunto(s)

Inmunosupresores/farmacología , Linfocitos/efectos de los fármacos , Bazo/efectos de los fármacos , Timo/efectos de los fármacos , Animales , Formación de Anticuerpos/efectos de los fármacos , Corticosterona/sangre , Corticosterona/farmacología , Ciclofosfamida/farmacología , Citarabina/farmacología , Femenino , Técnica del Anticuerpo Fluorescente , Reacción Injerto-Huésped/efectos de los fármacos , Hidrocortisona/farmacología , Linfocitos/clasificación , Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Pirimidinonas/farmacología , Bazo/citología , Bazo/inmunología , Timo/citología , Timo/inmunología

RESUMEN

We have used spin labeling, fluorescence polarization, and chemical analysis to characterize membrane properties of thymocytes from mice treated with immunomodulatory drugs. The number of thymocytes was reduced 90-95% by treatment of 6-9 week old mice with hydrocortisone acetate (HCA) or methylprednisolone (both 125 mg/kg) or with cyclophosphamide (250 mg/kg). Electron spin resonance (esr) examination of thymocytes labeled with 5-nitroxyl stearic acid indicated that the membranes of cells remaining after treatment with any of these drugs were more rigid than those from saline-treated controls. The total cholesterol/phospholipid (C/PL) molar ratio of the HCA-resistant thymocytes was twice that of the control mice. Treatment of mice with other immunomodulatory drugs, cyclophosphamide, cytosine arabinoside (Ara-C), 2-amino-5-bromo-6-phenyl-4-(3H)-pyrimidinone (ABPP) and 15(S)-methyl prostaglandin E1 (15(S)-methyl PGE1), also altered the C/PL ratio in thymocytes and, in some cases, in spleen cells. Fluorescence polarization measurements of thymocytes labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) did not reveal the differences between cells from HCA-and saline-treated mice that were detected by spin labeling and chemical analysis. Our results indicate that the greater rigidity detected by spin labeling of hydrocortisone-resistant thymocytes may be due, at least in part, to greater membrane cholesterol content. Of the methods employed, chemical analysis was the most sensitive in revealing drug-induced alterations in thymocyte populations.

Asunto(s)

Alprostadil/análogos & derivados , Colesterol/análisis , Inmunosupresores/farmacología , Bazo/efectos de los fármacos , Timo/efectos de los fármacos , Animales , Membrana Celular/análisis , Membrana Celular/efectos de los fármacos , Ciclofosfamida/farmacología , Citarabina/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Femenino , Hidrocortisona/análogos & derivados , Hidrocortisona/farmacología , Masculino , Metilprednisolona/farmacología , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Prostaglandinas E Sintéticas , Pirimidinas/farmacología , Espectrometría de Fluorescencia , Bazo/análisis , Bazo/citología , Timo/análisis , Timo/citología