RESUMEN
The results of many different types of animal and human studies dealing with the biological effects of exposure to low frequency electromagnetic fields (EMFs) have consistently been both positive and negative. We addressed the question of why this pattern had occurred so commonly in biological studies involving exposure to EMFs and hypothesized that it stemmed from the prevalent use of a linear model to characterize what are inherently nonlinear input-output relationships. The hypothesis was tested by analyzing biological data using a novel statistical procedure that could be adjusted to detect either nonlinear or linear effects. The reliability of the procedure was established using positive and negative controls and by comparison with the results obtained from sampling a known nonlinear system. In four independent experiments, male and female mice were exposed continuously to 0.1 or 0.5 mT, 60 Hz, for 175 days, and the effect on 20 immune parameters was measured using flow cytometry and functional assays. In each experiment, EMF exposure resulted in statistically significant changes in lymphoid phenotype when and only when the response of the animals to the fields was analyzed as if it were governed by nonlinear laws. Our results suggest that the pattern of inconsistency in the EMF bioeffects studies is an artifact resulting from an incorrect choice of the conceptual model for the relation between the field and the biological effect it causally determines.
Asunto(s)
Campos Electromagnéticos/efectos adversos , Linfocitos/inmunología , Animales , Antígenos CD/metabolismo , Fenómenos Biofísicos , Biofisica , Células de la Médula Ósea/inmunología , Femenino , Humanos , Inmunoglobulinas/metabolismo , Funciones de Verosimilitud , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Dinámicas no Lineales , Bazo/citología , Bazo/inmunología , Timo/citología , Timo/inmunologíaRESUMEN
OBJECTIVE: The characteristic biological effects of low-frequency electromagnetic fields (EMFs) appear to be functional changes in the central nervous, endocrine and immune systems. For unapparent reasons, however, the results of similar studies have often differed markedly from one another. We recognized that it had generally been assumed, in the studies, that EMF effects would exhibit a dose-effect relationship, which is a basic property of linear systems. Prompted by recent developments in the theory on nonlinear systems, we hypothesized that there was a nonlinear relationship between EMFs and the effects they produced in the endocrine and immune systems. METHODS: We developed a novel analytical method that could be used to distinguish between linear and nonlinear effects, and we employed it to examine the effect of EMFs on the endocrine and immune systems. RESULTS: Mice exposed to 5 G, 60 Hz for 1-175 days in 7 independent experiments reliably exhibited changes in serum corticosterone and lymphoid phenotype when the data were analyzed while allowing that the field exposure and the resulting effects could be nonlinearly related. When the analysis was restricted to linear relationships, no effects due to the field were found. CONCLUSIONS: The results indicated that transduction of EMFs resulted in changes in both the endocrine and immune systems, and that the laws governing the changes in each system were not the type that govern conventional dose-effect relationships. Evidence based on mathematical modeling was found suggesting that the coincident changes could have been causally related.
Asunto(s)
Campos Electromagnéticos , Sistema Endocrino/efectos de la radiación , Sistema Inmunológico/efectos de la radiación , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/efectos de la radiación , Corticosterona/sangre , Relación Dosis-Respuesta en la Radiación , Sistema Inmunológico/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Neuroinmunomodulación/efectos de la radiación , Dinámicas no Lineales , Bazo/inmunología , Bazo/efectos de la radiación , Timo/inmunología , Timo/efectos de la radiaciónRESUMEN
Animal studies of the effects of low-frequency electromagnetic fields (EMFs) on the immune system appear inconsistent, and recent evidence indicates that inconspicuous experimental problems are not responsible. We hypothesized that the inconsistencies resulted from use of linear methods and models to study inherently nonlinear input-output relationships. Using a novel analytical method, we found that exposure of mice to 5 G, 60 Hz, for 1-105 days in 6 independent experiments consistently affected a broad panel of immune variables when and only when the reaction of the immune system was modeled to allow the possibility of nonlinearity in the relationship between the field and the immune variables. It was possible to mimic the pattern observed in the immune data by sampling from a known chaotic system, suggesting the possibility that the observed pattern was the result of intrinsic nonlinear regulatory mechanisms in the immune system. Overall, the results suggested that lymphoid sub-populations were vulnerable to the physiological consequences of EMF transduction, that it may never be possible to predict specific changes in particular immune-system variables, and that the underlying behavior of the immune system (that which occurs in the absence of specific inputs) may be governed by laws that manifest extreme sensitivity to prior states.
Asunto(s)
Células de la Médula Ósea/citología , Campos Electromagnéticos/efectos adversos , Bazo/citología , Timo/citología , Animales , Femenino , Sistema Inmunológico , Subgrupos Linfocitarios , Ratones , Ratones Endogámicos C57BL , Dinámicas no LinealesRESUMEN
The neurochemical and endocrine responses to inoculation of mice with the murine lymphoma cell line AW5E was studied. This cell line was chosen because it is NK cell lysis resistant and thus does not induce a normal immune response. Immune activation has long been known to be a potent stimulator of the hypothalamo-pituitary-adrenocortical (HPA) axis as well as brain catecholamine and indoleamine metabolism, involving increases in the brain concentrations of catabolites of norepinephrine (NE) and serotonin (5-HT), as well as free tryptophan. Mice injected intravenously with AW5E tumor cells exhibited small increases in plasma corticosterone and hypothalamic NE and 5-HT catabolites one day after injection. There were no significant changes after 6 or 8 days, but a sustained increase in hypothalamic NE and 5-HT metabolism appeared 10 days after injection. There were similar, but more limited changes in the brain stem and prefrontal cortex. On the last day tested (day 14), plasma corticosterone was slightly elevated, as were hypothalamic dopamine, NE and 5-HT catabolites and tryptophan. These results indicate that inoculation with AW5E tumor cells increases brain catecholamine and serotonin metabolism, the hypothalamus being the most sensitive region. The most marked increases occurred in the few days preceding death, and thus may be associated with the pathology of the tumor growth.
Asunto(s)
Corticoesteroides/metabolismo , Monoaminas Biogénicas/metabolismo , Catecolaminas/metabolismo , Linfoma/metabolismo , Corticoesteroides/sangre , Animales , Química Encefálica , Tronco Encefálico/metabolismo , Dopamina/metabolismo , Hipotálamo/metabolismo , Cinética , Masculino , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Corteza Prefrontal/metabolismo , Serotonina/metabolismo , Triptófano/metabolismo , Células Tumorales CultivadasRESUMEN
Studies of the effects of power-frequency electromagnetic fields (EMFs) on the immune and other body systems produced positive and negative results, and this pattern was usually interpreted to indicate the absence of real effects. However, if the biological effects of EMFs were governed by nonlinear laws, deterministic responses to fields could occur that were both real and inconsistent, thereby leading to both types of results. The hypothesis of real inconsistent effects due to EMFs was tested by exposing mice to 1 G, 60 Hz for 1-105 days and observing the effect on 20 immune parameters, using flow cytometry and functional assays. The data were evaluated by means of a novel statistical procedure that avoided averaging away oppositely directed changes in different animals, which we perceived to be the problem in some of the earlier EMF studies. The reliability of the procedure was shown using appropriate controls. In three independent experiments involving exposure for 21 or more days, the field altered lymphoid phenotype even though the changes in individual immune parameters were inconsistent. When the data were evaluated using traditional linear statistical methods, no significant difference in any immune parameter was found. We were able to mimic the results by sampling from known chaotic systems, suggesting that deterministic chaos could explain the effect of fields on the immune system. We conclude that exposure to power-frequency fields produced changes in the immune system that were both real and inconsistent.
Asunto(s)
Campos Electromagnéticos , Sistema Inmunológico/fisiología , Sistema Inmunológico/efectos de la radiación , Dinámicas no Lineales , Animales , Biomarcadores , Células de la Médula Ósea/citología , Células Cultivadas , Radioisótopos de Cromo , Relación Dosis-Respuesta Inmunológica , Relación Dosis-Respuesta en la Radiación , Suministros de Energía Eléctrica , Exposición a Riesgos Ambientales , Femenino , Citometría de Flujo , Sistema Inmunológico/citología , Subgrupos Linfocitarios/citología , Subgrupos Linfocitarios/fisiología , Subgrupos Linfocitarios/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos C57BL , Bazo/citología , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/fisiología , Timo/citologíaRESUMEN
Optimal immunological control of cutaneous herpes simplex virus type 1 (HSV-1) infections initiated in the hind footpad of C57BL/6 (B6, H-2b) mice is dependent upon the presence of functional HSV-1-specific T lymphocytes. The class I MHC-restricted, CD8+ T cell subpopulation is involved in the clearance of infectious HSV-1 from the skin and limiting HSV-1 replication and spread within the peripheral nervous system. However, the frequency of HSV-1-specific CTL precursors (CTLp), as a measure of potential anti-viral CD8+ T cell function, is relatively low compared with other acute viral infections. To gain insight into the basis for this low functional frequency, changes in the CD8+ T cell subpopulation phenotype associated with activation and differentiation were investigated. Analysis of the phenotypic changes showed that HSV-1-specific CTLp were found predominantly within a subpopulation of CD8+ T cells expressing high levels of CD44 (CD44high) and high levels of the IL-2 receptor alpha-chain (CD25high). A second activated subpopulation of CD8+ T cells expressing the CD44high CD25low phenotype did not contain detectable HSV-1-specific CTLp, even after the addition of HSV-1-infected stimulator cells as a source of an exogenous Ag. These data suggested that HSV-1-specific CD8+ T cells must increase expression of CD25 before attaining the potential to become CTL effector cells. These findings also indicated that the up-regulation of CD44 alone is not sufficient to identify precisely HSV-1-specific CD8+ T cells.
Asunto(s)
Herpes Simple/inmunología , Herpesvirus Humano 1/inmunología , Ganglios Linfáticos/inmunología , Células Madre/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Enfermedad Aguda , Animales , Línea Celular , Chlorocebus aethiops , Citotoxicidad Inmunológica , Herpes Simple/patología , Receptores de Hialuranos/biosíntesis , Inmunofenotipificación , Ganglios Linfáticos/patología , Ganglios Linfáticos/virología , Recuento de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Interleucina-2/análisis , Células Madre/patología , Células Madre/virología , Subgrupos de Linfocitos T/patología , Subgrupos de Linfocitos T/virología , Linfocitos T Citotóxicos/patología , Linfocitos T Citotóxicos/virología , Regulación hacia Arriba/inmunologíaRESUMEN
The effects of in utero alcohol exposure on neonatal lymphopoiesis were examined in a murine model of fetal alcohol syndrome. At birth, both immature and mature B cells were decreased in the spleens of neonatal animals and these subpopulations of B cells did not recover to normal levels until 3-4 weeks of life. Pre-B cells and total B cells were decreased as well in the bone marrow of ethanol-exposed animals. By 3-4 weeks of life, the number of B cells in the bone marrow recovered to normal levels, but the pre-B cells remained below normal levels through 5 weeks of age. Furthermore, a recently described early B cell progenitor was reduced in frequency in ethanol-exposed neonates. Together, these data suggest that in utero exposure to ethanol can result in abnormalities in B cell development that may initiate at an early stage of B cell development.
Asunto(s)
Linfocitos B/inmunología , Células de la Médula Ósea/inmunología , Trastornos del Espectro Alcohólico Fetal/inmunología , Hematopoyesis , Bazo/inmunología , Animales , Animales Recién Nacidos , Linfocitos B/citología , Biomarcadores , Células de la Médula Ósea/citología , Linaje de la Célula , Femenino , Células Madre Hematopoyéticas , Ratones , Embarazo , Bazo/citologíaRESUMEN
Fetal alcohol syndrome is one of the leading causes of birth defects in this country. Children exposed to alcohol in utero suffer from growth and mental retardation, physical abnormalities, and immune dysfunction. Previous work from this laboratory demonstrated that B lymphopoiesis is delayed in mice exposed to alcohol in utero. The deficit in B-cell development was apparent shortly after birth and extended to well after weaning. Because lymphopoiesis begins in the fetal liver, the current study was done to determine if fetal B-cell development was affected as well by in utero exposure to alcohol. We now show that the effects of in utero alcohol exposure on B lymphopoiesis do not become apparent until late in gestation. Flow cytometry was used to enumerate several intermediates in the B-cell developmental pathway. These phenotypic analyses showed that before day 17 of gestation, B-lineage intermediates developed normally when compared with control animals. However, between days 17 and 18 of gestation, an abnormality in the population dynamics of B-lineage intermediates became apparent in the fetal liver of alcohol-exposed mice. Early intermediates in the B-cell developmental pathway were present in normal numbers; however, the more mature progenitors as well as B cells were decreased in number by gestational day 18. These data suggest that in utero alcohol exposure disrupts the ability of B-lineage intermediates to progress along the developmental pathway to maturity, thereby leaving the animal immunocompromised at birth.
Asunto(s)
Linfocitos B/efectos de los fármacos , Etanol/toxicidad , Trastornos del Espectro Alcohólico Fetal/inmunología , Hígado/efectos de los fármacos , Animales , Linfocitos B/inmunología , Femenino , Inmunocompetencia/efectos de los fármacos , Inmunocompetencia/inmunología , Hígado/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
The neonatal period marks an important time in mammalian immunologic development, yet it is often ignored in studies of lymphocyte development. We identified a cell population with the phenotype heat stable Ag (HSA)low lin- CD43low that contained B cell progenitors at a high frequency in the neonatal bone marrow and spleen. Although cells with a similar phenotype can be identified in the bone marrow and spleen of adult animals, these populations showed a greatly reduced frequency of B cell progenitors. B lineage cells were detected after 7 days in culture at a frequency of 1:15 when HSAlow lin- CD43low cells from neonatal bone marrow were cultured on stromal cells and IL-7 under limiting dilution conditions. Under similar conditions, the equivalent population in adult bone marrow had a frequency of B cell progenitors that was less than 1:2000. The expression of terminal deoxynucleotidyl transferase in freshly sorted neonatal HSAlow lin- CD43low cells suggested that cells committed to the lymphocyte lineage were present in this population. These data suggested that the HSAlow lin- CD43low population of cells represents a pool of B lineage precursors that may be responsible for filling the immune compartment early in neonatal life.
Asunto(s)
Envejecimiento/inmunología , Animales Recién Nacidos/inmunología , Subgrupos de Linfocitos B/inmunología , Células de la Médula Ósea/inmunología , Glicoproteínas de Membrana , Bazo/inmunología , Células Madre/inmunología , Animales , Antígenos CD/biosíntesis , Subgrupos de Linfocitos B/enzimología , Células de la Médula Ósea/enzimología , Antígeno CD24 , Diferenciación Celular/inmunología , División Celular/inmunología , Linaje de la Célula/inmunología , ADN Nucleotidilexotransferasa/biosíntesis , Femenino , Inmunofenotipificación , Leucosialina , Recuento de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Sialoglicoproteínas/biosíntesis , Bazo/citología , Bazo/enzimología , Células Madre/enzimologíaRESUMEN
Dose-response experiments were performed to establish an optimum concentration of ethanol (EtOH) in liquid diet formulations for use with a murine model (C57B1/6) of potential biological effects attributable to EtOH consumption. An optimum concentration was predetermined to be the highest EtOH concentration consumed by mice without resulting in a loss of body weight. Feeding trials were performed using EtOH concentrations that ranged from 25 to 36% ethanol-derived calories (EDC) during 7-day experiments, or 10 to 30% EDC fed during 21-day experiments. The parameters studied included body weight changes, diet consumptions, daily g EtOH kg-1 body weight, as well as differences in mononuclear cell numbers from the spleen, thymus, and bone marrow. Diet consumptions by the EtOH groups and pair-fed (PF) groups were monitored by weight rather than by volume. During either 7-day or 21-day trials, diet consumptions were lower by groups receiving diets of higher EtOH concentrations; however, daily EtOH intake was maximal by groups fed diets of 25% EDC in all experiments. These mice also gained weight, whereas mice maintained on 30% EDC did not gain weight, and mice maintained on diets of 33 or 36% EDC lost significant body weight. Body weight changes in PF groups were similar to their respective EtOH group. Changes in mononuclear cell numbers of the spleen and thymus paralleled the changes seen in body weights. In the 7-day trials, cell counts declined progressively in groups maintained on diets of high EDC (> or = 30% EDC) or their PF controls. From the 21-day trials, cell counts of both the 30% EDC group and their PF controls declined, compared with all other groups. Together, the conclusion drawn from these findings was that nutritional stress was principally responsible for the mononuclear cell depletions. This contradicts previous reports and highlights the need for strict attention to the pairfeeding paradigm to avoid masking a nutritional component of such studies through overfeeding of the PF controls. Liquid diets of 25% EDC were determined to be optimal for immunological studies using a murine model, because this concentration maximizes EtOH consumption and maintains body weight of the experimental animals.
Asunto(s)
Consumo de Bebidas Alcohólicas/efectos adversos , Modelos Animales de Enfermedad , Animales , Peso Corporal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Etanol/administración & dosificación , Etanol/toxicidad , Femenino , Recuento de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Necesidades NutricionalesRESUMEN
Rearrangement of the T cell antigen receptor genes is a complex, highly regulated process. To gain a better understanding of the extracellular factors involved in the regulation of TCR beta and gamma gene rearrangement in adult murine bone marrow-resident precursor T cells, several cytokines were tested for their ability to induce gene recombination. A selected population of C58/J bone marrow cells (Thy 1(low), CD3, CD8, B220) that is enriched for pre-T cell activity was propagated in vitro in medium supplemented with IL-3 and mast cell growth factor (MGF, also referred to as stem cell factor, Steele factor and c-kit ligand). These cytokines were required for the maintenance of pre-T cell activity in culture, but had no effect on TCR gene expression. Several additional cytokines were added to the culture medium. Of all those tested, only IL-7 induced complete rearrangement of the TCR gamma locus. Complete rearrangement of the TCR beta locus was not induced under any of the culture conditions analysed here. The bone marrow cells cultured in IL-3, MGF and IL-7 did not begin to express mature T cell proteins and maintained their in vivo progenitor potential. Furthermore, IL-7 cultured bone marrow cells were capable of differentiation in vivo into all phenotypic subpopulations of T cells, without an apparent bias toward the gammadelta lineage. The data presented here suggest that TCR gamma gene rearrangement in adult pre-T cells is regulated by IL-7, but that the TCR beta locus requires additional or alternative signals for the induction of complete rearrangement.
Asunto(s)
Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T/efectos de los fármacos , Reordenamiento Génico de la Cadena gamma de los Receptores de Antígenos de los Linfocitos T/efectos de los fármacos , Interleucina-7/farmacología , Linfocitos T/inmunología , Animales , Células de la Médula Ósea , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Inmunofenotipificación , Interleucina-3/farmacología , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos C57BL , ARN Mensajero/genética , Factor de Células Madre/farmacología , Transcripción GenéticaRESUMEN
The immune response to HSV infection in C57BL/6 mice includes a CTL population that recognizes the virion envelope glycoprotein gB. However, previous studies showed that CTL specific for other viral determinants were also responding to HSV infection. These studies demonstrate that an additional determinant is the HSV immediate-early protein ICP27. During the primary response, both gB- and ICP27-specific CTL were detected in the draining lymph node. In response to reinfection, ICP27-specific CTL were present early in the lymph node, while the appearance of gB-specific CTL activity was delayed. Analysis of the primary amino acid sequence of ICP27 predicted two potential Kb-binding epitopes, one of which sensitized uninfected cells for lysis by HSV-specific CTL. In addition, ICP27 epitope-specific CTL activity was detected in the splenic memory CTL pool. These results show that CTL which recognize different antigens may also exhibit differences in how they respond to HSV reinfection in vivo.
Asunto(s)
Herpes Simple/inmunología , Proteínas Inmediatas-Precoces , Memoria Inmunológica/inmunología , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Animales , Línea Celular , Chlorocebus aethiops , Epítopos/inmunología , Inmunización , Inmunización Secundaria , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Bazo/citología , Células Vero , Proteínas Virales/inmunologíaRESUMEN
The effect of intrauterine exposure to ethanol on lymphocyte development in the neonatal period was studied in C57BI/6J mice. Mice were bred, and then the female mice were assigned to 1 of 3 diet groups, 25% ethanol-derived calories (EDC), pair-fed control, or ad libitum laboratory chow. At birth, all offspring were cross-fostered to surrogate mothers who had been fed laboratory chow. At weekly intervals, the neonatal mice were weighed, and 4 mice from each group were used to assess the development of splenic lymphocytes. The total number of splenocytes was similar in all three groups at each sampling. The number of T-cells, B-cells, and natural killer (NK) cells was measured by flow cytometry. T-cells and NK cells did not vary significantly among the three diet groups. However, the total number of B-cells was decreased for the first 3 weeks of life in the ethanol-exposed animals. The function of the T-cells and B-cells was determined by assessing the response to lipopolysaccharide, pokeweed mitogen, phytohemagglutinin, and concanavalin A. The response to all four mitogens was significantly reduced in the ethanol-exposed animals and did not recover to control levels until 4-5 weeks of life. Ethanol exposure had no significant effect on the kinetics of acquisition of NK lytic function, as assessed by determining the killing of chromium-51 labeled YAC-1 tumor target cells. These data show that prenatal exposure to ethanol causes a transient immunodeficiency in some, but not all compartments of the immune system.
Asunto(s)
Linfocitos B/inmunología , Trastornos del Espectro Alcohólico Fetal/inmunología , Células Asesinas Naturales/inmunología , Linfocitos T/inmunología , Animales , Animales Recién Nacidos/inmunología , Línea Celular , Citotoxicidad Inmunológica/inmunología , Femenino , Tolerancia Inmunológica/fisiología , Leucemia Experimental , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Masculino , Ratones , EmbarazoRESUMEN
The effects of acute administration of ethanol (EtOH) on natural killer (NK) cells have not previously been examined in mice or humans. In the present study, a single dose of EtOH (5.0-7.0 g/kg) was administered by gavage to B6C3F1 mice. This produced maximum blood EtOH levels of approximately 0.25 to 0.50% (wt/vol). A single dose of EtOH decreased splenic NK cell activity (as measured by lysis of YAC-1 target cells in vitro). This decrease was maximal 12 hr after dosing and was no longer evident at 60 hr. Suppression of NK cell activity was consistently significant at EtOH doses of 6.0 or 6.5 g/kg, and significant suppression occurred in two of three experiments at doses of 5.0 or 5.5 g/kg. Flow cytometric analysis indicated a decrease in the percentage of NK cells in the spleen in EtOH-treated mice, and there was a small decrease in the total number of splenocytes. However, the decrease in the percentage of NK cells was significantly less than the decrease in NK cell activity, suggesting an effect on NK cell activity as well as NK cell number. Splenic T cells were not depleted, but B cells were significantly decreased at the highest EtOH dose. Enhancement of NK activity after in vivo administration of polyinosinic-polycytidilic acid was blocked by EtOH (6.0 g/kg). These results indicate acute exposure to EtOH decreases basal and induced splenic NK cell activity in mice and that loss of NK cells at least partially explains the decrease in basal NK cell activity.
Asunto(s)
Consumo de Bebidas Alcohólicas/sangre , Etanol/farmacología , Células Asesinas Naturales/efectos de los fármacos , Animales , Antígenos/análisis , Antígenos de Superficie , Relación Dosis-Respuesta a Droga , Etanol/sangre , Femenino , Citometría de Flujo , Lectinas Tipo C , Subgrupos Linfocitarios/efectos de los fármacos , Ratones , Subfamilia B de Receptores Similares a Lectina de Células NK , Poli I-C/farmacología , Proteínas/análisis , Bazo/citologíaRESUMEN
During their development, T-cell precursors (pre-T cells) undergo a variety of changes with respect to their expression of specific surface proteins. Among the most critical of the surface markers acquired by developing T cells is the T-cell receptor (TCR)/CD3 complex. Prior to the assembly and transport of complete TCR/CD3 multimeric complexes to the plasma membrane, the individual constituent subunits are expressed in the cytoplasm (ER-Golgi). In order to study the expression of the T-cell receptor TCR/CD3 complex during pre-thymic T-cell differentiation, we have developed a flow cytometric technique for the simultaneous detection of surface (sCD3 epsilon) and cytoplasmic CD3 epsilon (cCD3 epsilon). This technique, which employs fixation in 1% paraformaldehyde and permeabilization with 1% saponin and 0.025% digitonin, features reliable internalization and low nonspecific binding of anti-CD3 epsilon in murine lymphoid cells, as well as tissue culture cell lines. The combination of saponin and digitonin treatment was also compatible with the staining of sCD3 and other lymphocyte surface antigens such as Thy1, CD4, CD8, B220, and IgM. In contrast, permeabilization of cells with the detergents Tween 20 and Triton X-100 was shown to remove surface-bound anti-CD3 epsilon. The present technique permitted the detection of discernible sCD3 epsilon and cCD3 epsilon double and single positive lymphocytes and may prove useful in defining bone marrow-resident pre-T cells.
Asunto(s)
Complejo CD3/análisis , Citoplasma/inmunología , Citometría de Flujo/métodos , Linfocitos T/inmunología , Animales , Antígenos de Diferenciación de Linfocitos T/análisis , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Cultivadas , Digitonina/farmacología , Fijadores , Formaldehído , Masculino , Ratones , Ratones Endogámicos , Polímeros , Saponinas/farmacología , Bazo/citología , Timo/citologíaRESUMEN
The immune response to herpes simplex virus type 1 (HSV-1) infection in C57BL/6 mice includes a population of major histocompatibility complex class I-restricted cytolytic T lymphocytes (CTL) that recognize the structural glycoprotein gB. To gain insight into the importance of this CTL subpopulation in vivo, gB-specific CTL present in the regional lymph nodes after a primary infection and after a reinfection of convalescent animals were analyzed. In a primary infection, gB-specific CTL precursors (CTLp) that recognized either a cell line constitutively expressing gB or cells pulsed with the optimal Kb-restricted gB epitope 498SSIEFARL505 were present at an estimated frequency of 1/12,000 compared with a frequency of 1/3,000 for CTLp which recognized cells infected with HSV-1 itself. In convalescent mice responding to reinfection, HSV-specific CTLp were present at an estimated frequency of 1/4,000 to 1/14,000. However, gB-specific CTLp could not be detected at this site. These findings suggest that CTL specific for an immunodominant epitope contribute substantially to the primary response but may not be a component of the HSV-specific CTL population that responds rapidly to reinfection in vivo.
Asunto(s)
Herpes Simple/inmunología , Herpesvirus Humano 1/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Animales , Células Madre Hematopoyéticas/inmunología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia MolecularRESUMEN
It is well established that T lymphocytes play a critical role in the control and clearance of herpes simplex virus (HSV) infections. However, the role of the CD4+ and CD8+ T cell subsets in the recovery process has not been clearly elucidated. Cutaneous HSV infection of the footpad tissue of C57BL/6 (B6) mice provides a model to determine the relative contribution of each T cell subset during the important early phase of the response to infection. In this study, we observed that the elimination of mature peripheral T lymphocytes by depletion in vivo with a combination of Cd4- and CD8-specific monoclonal antibodies prevented recovery from acute infection in this model. However, mice depleted of either the CD4+ or CD8+ subpopulation alone recovered completely, with only a slight delay in the total clearance of infectious virus. Adoptive transfer studies revealed that lymph node cells from donor mice selectively depleted of either CD4+ or CD8+ T cell subset in vivo, or from normal donors selectively depleted in vitro, were able to mediate recovery. As CD4-depleted mice fail to generate a CD8+ T cell-mediated cytolytic T lymphocyte (CTL) response, this suggested that the control of cutaneous HSV infections may be mediated by a cytokine-dependent mechanism common to both the CD4- and CD8+ T cell subpopulations. It was subsequently found that the neutralization of IFN-gamma in vivo diminished the ability of mice to clear infectious HSV from the skin, and treatment with anti-IFN-gamma in vivo ablated the ability of transferred T cells to mediate recovery. These studies suggested that IFN-gamma-mediated mechanisms play a critical role in the control of and recovery from acute cutaneous HSV infection.
Asunto(s)
Herpes Simple/inmunología , Interferón gamma/inmunología , Subgrupos de Linfocitos T/inmunología , Enfermedad Aguda , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Antígenos CD4 , Linfocitos T CD4-Positivos/inmunología , Antígenos CD8 , Línea Celular Transformada , Herpes Simple/microbiología , Inmunoterapia Adoptiva , Masculino , Ratones , Ratones Endogámicos C57BL , Pruebas de Neutralización , Simplexvirus , Piel/microbiología , Piel/efectos de la radiación , Linfocitos T Reguladores/inmunología , Células Vero , Replicación ViralRESUMEN
A very small fraction of thymocytes has recently been identified that expresses low levels of CD4 in the absence of CD8, CD3, or a TCR. These CD4lo thymocytes appear to be the precursors of the early CD4-CD8-CD3- thymic subset and contain most of the T cell progenitor activity found within the thymus. Here, we examined adult bone marrow for the presence of a similar population of cells and found that 0.5% to 3.5% of C58/J bone marrow cells express low, but detectable levels of CD4 (CD4lo) at the cell surface in the absence of CD3. These CD4lo bone marrow cells display pre-T cell activity, in that they are able to repopulate the thymus of irradiated recipient mice after intrathymic transfer. Moreover, we found that most of pre-T cell activity found in the bone marrow is contained within the CD4lo expressing subset of marrow cells. Although the CD4lo cells found in both the thymus and bone marrow display pre-T cell activity, the CD4lo cells from these two sites showed pronounced differences with respect to their ability to respond to specific cytokine stimulation in vitro. Bone marrow-resident CD4lo cells proliferated in response to both IL-3 and mast cell growth factor in vitro, whereas CD4lo cells isolated from the thymus did not. Furthermore, CD4lo bone marrow cells, grown in media containing IL-3 and mast cell growth factor, retained their pre-T cell activity, indicating that CD4lo cells with pre-T cell capabilities were among the IL-3 and mast cell growth factor-responsive cells. These data suggest that although pre-T cells in bone marrow share the CD4lo phenotype with their intrathymic counterparts, they may be fundamentally different with respect to the environmental factors that control their growth.
Asunto(s)
Células de la Médula Ósea , Antígenos CD4/análisis , Células Madre Hematopoyéticas/inmunología , Linfocitos T/inmunología , Timo/citología , Animales , Células Cultivadas , Infecciones por VIH/inmunología , Factores de Crecimiento de Célula Hematopoyética/farmacología , Interleucina-3/farmacología , Masculino , Ratones , Ratones Endogámicos AKR , Factor de Células MadreRESUMEN
We have utilized several clonal cell lines, derived from the murine lymphoma ASL1w, to investigate the early events in NK-mediated lysis. The studies described here examine the relationship between NK recognition, NK cell:tumor cell conjugate formation, and NK-mediated lysis. The AW4F and AW4D tumor lines were susceptible to NK-mediated lysis and efficiently inhibited NK recognition in competitive inhibition assays, whereas the AW5J tumor, which is relatively resistant to NK-mediated lysis, did not. In contrast, the AW5E tumor was NK resistant but inhibited NK recognition almost as well as the NK-sensitive tumors, suggesting that it was deficient in a postbinding event required for NK-mediated lysis. These findings demonstrate a correlation, with one exception, between the susceptibility of the ASL1w-derived tumor lines to NK-mediated lysis and their ability to inhibit NK recognition. In contrast, there was no apparent correlation between tight conjugate formation, as assessed in three independent target binding assays, and the susceptibility of these tumors to NK-mediated lysis, showing that tight conjugate formation is not required for either efficient NK recognition or lysis.
Asunto(s)
Comunicación Celular , Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Animales , Linfoma/patología , Masculino , Ratones , Ratones Endogámicos C3H , Bazo/inmunología , Células Tumorales CultivadasRESUMEN
We have developed a model system which can be utilized for characterizing both the molecular basis of natural killer (NK):tumor interactions and the consequences of these interactions on tumor growth in vivo. This model system consists of several tumor lines which were derived from the murine lymphoma ASL1w under conditions which permitted the isolation of clonal lines that differ in their susceptibility to NK-mediated lysis. The NK-resistant clones used in this study (AW5J and AW5E) were susceptible to cytotoxic T-lymphocyte-mediated lysis and thus appear to be resistant to lytic processes utilized uniquely by NK cells. In competitive (cold target) inhibition assays, the AW5J clone did not inhibit NK recognition as well as the NK-sensitive clones. Thus AW5J may not be efficiently recognized by NK cells. The AW5E clone, on the other hand, competitively inhibited NK recognition as efficiently as the NK-sensitive clones; therefore, AW5E appears to evade a post-recognition event which is required for NK-mediated lysis. The susceptibility of these clones to killing by NK cells directly correlated with their lethality, suggesting that NK cells regulated the growth of these tumors in vivo. The level of host NK activity was also an important determinant of the level and site of tumor localization. Two-step immunofluorescence assays and flow cytometric analysis were used to quantitate the number of tumor cells in the bone marrow, spleen, and lymph nodes of mice with augmented or depleted NK activity. Increasing host NK activity decreased the number of tumor cells in each organ which could support the growth of the particular tumor tested. Furthermore, the extent of NK regulation was dependent, in part, upon the susceptibility of the particular tumor to NK-mediated lysis and the site of tumor growth. Thus, the data presented here demonstrate the utility of the ASL1w-derived clones as a model system which can be used to delineate the requirements and consequences of NK:tumor interactions.