RESUMEN
Investigation of the biological actions of loxoribine in chronic lymphocytic leukemia (CLL) was undertaken because of the pervasive immunostimulatory effects of the nucleoside on normal B cells. In vitro studies with cells from a spectrum of CLL patients demonstrate that loxoribine induces B-CLL cells to enter and traverse the cell cycle. This is reflected by marked increases in DNA synthesis, by standard morphological criteria, and by flow cytometric evaluation of cell cycle status and of cell surface activation markers. Cells from about 75% of patients studied evince this response. Analysis of a variety of biological parameters indicate that only the ratio of T cells (CD4+ or CD8+) to B-CLL cells correlates with induction and degree of proliferative response. Co-stimulation with loxoribine and IL-2 results in modest proliferative synergy, presumably due to upregulation of IL-2R alpha expression on B-CLL cells by loxoribine. Prolonged exposure of B-CLL cells to stimulatory concentrations of loxoribine frequently culminates in progression of the responsive cells to apoptosis. The capacity of loxoribine to transiently approximate the reversible transformation of a low grade B cell malignancy to one of a higher grade presents the opportunity for evaluation of cycle-active drugs under these conditions. Recent studies indicate that pre-treatment of B-CLL cells with loxoribine results in synergistic killing of leukemic cells with cycle-active drugs. The ability to induce B-CLL cells into cell cycle entry and/or into either activation-induced apoptosis or into phases of the cell cycle sensitive to cytotoxic therapy opens up new perspectives for the development of potentially curative strategies for this chronic leukemia.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Linfocitos B/efectos de los fármacos , Guanosina/análogos & derivados , Leucemia Linfocítica Crónica de Células B/terapia , Adyuvantes Inmunológicos/farmacología , Apoptosis/efectos de los fármacos , Linfocitos B/patología , Ciclo Celular/efectos de los fármacos , Sinergismo Farmacológico , Guanosina/farmacología , Guanosina/uso terapéutico , Humanos , Inmunofenotipificación , Interleucina-2/farmacología , Interleucina-2/uso terapéutico , Leucemia Linfocítica Crónica de Células B/inmunología , Activación de Linfocitos/efectos de los fármacos , Recuento de Linfocitos/efectos de los fármacos , Receptores de Interleucina-2/biosíntesis , Receptores de Interleucina-2/genética , Subgrupos de Linfocitos T , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
To optimally modulate a system as complex as the immune system, one must ultimately control its elements individually. Up to this time, use of polyclonal immune stimulants has necessarily involved modulation of a block of immune functions, frequently including undesired activities as well as the activity of interest. We now report selective modulation of individual elements of the immune system by low molecular weight nucleosides, within the context of a fully functional immune system. Loxoribine (7-allyl-8-oxoguanosine) is a well characterized pleiotropic agonist of the immune system in a variety of species, including mouse and humans. In B-cells it binds to soluble cytoplasmic binding proteins, which upregulate transcription upon translocation to the nucleus. By altering specific portions of the loxoribine molecule, multiple distinct, bioactivity profiles have now been obtained. These include: 1) selective augmentation of antibody responses without effects on B-cell proliferation or NK-cell activity; 2) selective enhancement of NK-cell activity and B-cell proliferation in the absence of antibody responses; and 3) selective enhancement of NK-cell activity and antibody responses without B-cell proliferation. Predominant NK-cell responses with minimal B-cell activity of either type also can be generated. The pattern of cytokine mRNA transcription induced is consistent with the spectrum of cellular activities observed. Thus, it is possible to modulate selective activities of the immune system by relatively minor structural modifications of a broad-spectrum immunomodulator in an unseparated cell system.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Guanosina/análogos & derivados , Sistema Inmunológico/efectos de los fármacos , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Células Cultivadas , Citocinas/biosíntesis , Citocinas/genética , Citotoxicidad Inmunológica/efectos de los fármacos , Citotoxicidad Inmunológica/inmunología , Guanosina/farmacología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos , Peso Molecular , ARN Mensajero/genética , Bazo/citología , Bazo/inmunologíaRESUMEN
C8- and N7, C8-substituted guanine ribonucleosides comprise a class of molecules with potent immunostimulatory activity for a variety of humoral and cellular immune responses. Although it has been suggested that the immunostimulatory activity may be partially mediated by cytokine production, to date there has been no systematic evaluation of the spectrum of cytokines elicited by these nucleosides. In this study, we examine the cytokines produced by murine spleen cells in response to the di-substituted guanosine analog loxoribine (7-allyl-8-oxoguanosine). First, the levels of cytokine mRNA in spleens from vehicle- or loxoribine-treated mice were compared using PCR analysis with a panel of cytokine-specific primers. Enhancement of IL-1 alpha, TNF-alpha, TNF-beta, IL-6, IFN-alpha, and IFN-gamma mRNA was seen in the spleens of loxoribine-treated mice. IL-12 mRNA responses were more complex, with an increase in the p40 chain and a decrease in the p35 chain. In contrast, no increase was seen for mRNA levels of IL-2, IL-3, IL-4, IL-5, IL-7, or GM-CSF. ELISA assays on the supernatants of loxoribine-treated spleen cells demonstrated that IL-1 alpha, IL-6, TNF-alpha, and IFN-gamma were all produced in a dose-dependent fashion with TNF-alpha produced first, followed by IL-6 and IFN-gamma, and last by IL-1 alpha. IFN-alpha beta activity rose as quickly as TNF-alpha, leveling off at 8 to 12 hr, and was supplanted by a later-occurring surge of IFN-gamma production. IL-1 alpha, IL-6, TNF-alpha, and IFN-gamma were also detected in the sera of mice injected with loxoribine. When antibodies against the relevant cytokines were tested, only anti-IFN-alpha beta inhibited NK activity or lymphocyte proliferation and, in both cases, activity was partially restored by the addition of exogenous IFN-alpha beta. Taken together, these data indicate that loxoribine induces the production of a selective cohort of cytokines all of which have been shown to have immunostimulatory activity. However, only IFN-alpha beta appears to play a role in the enhancement of NK activity and lymphocyte proliferation.
Asunto(s)
Citocinas/biosíntesis , Guanosina/análogos & derivados , Animales , Secuencia de Bases , Citocinas/genética , Cartilla de ADN/química , Expresión Génica , Guanosina/farmacología , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos CBA , Datos de Secuencia Molecular , ARN Mensajero/genética , Bazo/metabolismoRESUMEN
A multiple immunodeficiency, involving antibody- and cell-mediated responses in 10 Chinese Shar-Pei (CSP) dogs is described. Abnormal levels of serum IgM and IgA in most cases, and IgG in fewer cases characterized the immunoglobulin deficiencies. Decreased in vitro proliferative responses of pokeweed mitogen (PWM)-stimulated peripheral blood mononuclear cells (PBMC) were found in nine cases. Clinical presentation involved several organ systems and was associated with recurrent infections and malignancy. Sera from affected dogs suppressed PWM-stimulated cell proliferation of affected and normal dogs, but not cultures stimulated with PWM followed by recombinant IL-2 (rIL-2). In vitro supplementation of PBMC cultures with immunomodulatory guanosine analogs (GA) resulted in increased de novo IgG and/or interleukin-6 (IL-6) synthesis. Cells from five immunodeficient dogs showed in vitro evidence of GA- or rIL-2-dependent enhanced immunological responses. Since rIL-2-mediated activation of the IL-2 receptor and GA-mediated immunomodulation are reported to act through protein kinase C (PKC)-independent pathways, it is concluded that the IL-2 receptor is functional in these dogs and that cell activation through alternative pathways may restore immune responses in affected CSP dogs.
Asunto(s)
Linfocitos B/inmunología , Enfermedades de los Perros/inmunología , Síndromes de Inmunodeficiencia/inmunología , Síndromes de Inmunodeficiencia/veterinaria , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Animales , Células Cultivadas , PerrosRESUMEN
Guanine ribonucleosides with single substitutions at the C8 position (monosubstituted) or with dual substitutions at the C8 and N7 positions (disubstituted) up-regulate a spectrum of immunologic responses, including cytolytic responses to tumor cells. The current studies were undertaken to determine the effects of dual substitution on a number of nucleoside-inducible immunological parameters. To do so, two monosubstituted analogues, 8-bromoguanosine and 8-mercaptoguanosine, were directly compared with two disubstituted analogues, 7-methyl-8-oxoguanosine and 7-allyl-8-oxoguanosine (loxoribine). All of the compounds enhance natural killer (NK) activity, lymphocyte proliferation, and antibody production in dose-dependent fashion. However, the potency and maximal activity of the disubstituted analogues are considerably greater than those of the monosubstituted analogues. Spleen cells stimulated for 48 h with the disubstituted compounds produce immunoreactive interleukin (IL) 1 alpha, IL-6, tumor necrosis factor-alpha (TNF alpha), and interferon-gamma (IFN gamma). Monosubstituted analogues induce lower quantities of IL-6, TNF alpha, and IFN gamma and fail to induce detectable levels of IL-1 alpha. Total IFN activity, assessed by viral inhibition assay, is also lower for the monosubstituted analogues. Augmentation of antibody secretion by B cells is diminished for neither mono- nor disubstituted compounds upon incubation with anti-cytokine antibodies. In contrast, anti-IFN alpha beta markedly reduces the effects of monosubstituted analogues on NK activity but has less marked effects on NK induction by the disubstituted compounds. A similar pattern of differences is seen for lymphocyte proliferation. Thus, although the analogues induce synthesis of several cytokines, to date only IFN alpha beta appears directly involved in enhancement of NK activity and lymphocyte proliferation. The present data do not, however, exclude the existence of an autocrine stimulatory mechanism not susceptible to inhibition by anti-cytokine antibodies.
Asunto(s)
Citocinas/biosíntesis , Guanosina/análogos & derivados , Guanosina/inmunología , Adyuvantes Inmunológicos/fisiología , Animales , Pruebas Inmunológicas de Citotoxicidad/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunidad Celular/fisiología , Inmunoglobulinas/biosíntesis , Técnicas Inmunológicas/normas , Interferón Tipo I/biosíntesis , Interferón gamma/análisis , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos CBA , Pruebas de Neutralización/métodos , Células Tumorales Cultivadas , Virus de la Estomatitis Vesicular Indiana/inmunologíaRESUMEN
Loxoribine is a potent new immunostimulant with a relatively broad spectrum of immunobiological activities. Both loxoribine and its analogues function as agonists of immune responses in a variety of species, including humans. They upregulate the activity of B cells, T cells, NK cells, macrophages, and LAK cells. Induction of enhanced cytokine secretion has been found to involve IFN-alpha/beta, IFN-gamma, TNF-alpha, TNF-beta, IL-1, IL-6, and the 40 kDa chain of IL-12. Evaluation of in vivo activity has been undertaken only for antibody production, NK cell-mediated cytotoxicity, induction of certain cytokines, and LAK cell-mediated cytotoxicity; all four types of activity are markedly upregulated by loxoribine in vivo. Augmentation of antibody production has been observed for protein, recombinant protein, and synthetic peptide antigens, among others. Because loxoribine and its analogues transmit a T-helper-like signal to antibody-producing B cells, it is a highly effective adjuvant even for synthetic peptides that lack T-cell epitopes, effectively replacing the function of T-helper cells in this milieu. It thus provides an alternative, T-cell-independent vaccination strategy if it becomes desirable to avoid untoward T-cell-mediated effects, or in patients with functional or absolute T-cell deficiency. There are a number of features unique to loxoribine that are highly advantageous under specific circumstances: (1) T cell independence; (2) loxoribine augments antibody responses from an intracellular location (rather than at the surface membrane), independently of protein kinase C involvement; this may be particularly relevant for patients with membrane receptor/signal transduction defects; (3) adjuvanticity of loxoribine is essentially free of cytokine dependency; this may be of particular value for organ transplantation patients whose cytokine-dependent immunity is pharmacologically suppressed; (4) loxoribine bypasses functional immunological immaturity, rendering it particularly useful for vaccines in infants. In preclinical safety studies, the drug has exhibited a relatively benign profile. Phase I clinical studies to date have produced no toxicity higher than grade 1. The drug appears to be quite stable, and compares very favorably in direct evaluations with a number of other immunostimulators. A number of clinical trials have been planned for the future.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Guanosina/análogos & derivados , Linfocitos T Colaboradores-Inductores/inmunología , Vacunas/inmunología , Adyuvantes Inmunológicos/efectos adversos , Adyuvantes Inmunológicos/uso terapéutico , Animales , Guanosina/efectos adversos , Guanosina/farmacología , Guanosina/uso terapéutico , Humanos , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Vacunas/administración & dosificación , Vacunas/efectos adversosRESUMEN
7-Allyl-8-oxoguanosine (loxoribine) is a di-substituted guanine ribonucleoside which has been shown previously to enhance murine NK activity, B lymphocyte proliferation, and antibody synthesis. In this study we examined the relationship among enhancement of NK activity, proliferation, and cytokine synthesis in the responses of different strains of mice to loxoribine to provide insight into the role of cytokines in these biological activities. The NK response of mice was enhanced both in vitro and in vivo in all strains tested with the exception of the NK-deficient beige (BgBg) mouse. However, there was a marked difference in the degree of NK enhancement noted in other inbred strains, with C3H and CBA mice producing the highest responses, C57BL/6, BALB/c, and DBA/2 strains giving intermediate responses, and SJL mice manifesting low responses. Striking enhancement of NK cell activity was seen in SCID mice. A somewhat different effect was seen in humans. Loxoribine treatment enhanced both the NK and LAK activity of cells from individuals with low and high spontaneous NK activity. The degree of enhancement was similar for both groups, and thus the general hierarchy of NK activity among different donors was maintained. There was less interstrain variation in the murine proliferative response to loxoribine although nude (NuNu) mice showed the highest activity and SJL mice produced substantially lower responses than other strains. All strains produced IL-6, TNF alpha, IFN-alpha/beta, and IFN-gamma when spleen cells were cultured for 48 hr with loxoribine. Interstrain variability of cytokine synthesis displayed no consistent pattern from one cytokine to another, and all failed to correlate with interstrain variability of NK cell activity or B lymphocyte proliferation. When anti-cytokine antibodies were tested for the ability to block the immunostimulatory effects of loxoribine, only anti-IFN-alpha/beta and, to a lesser degree, anti-IFN-beta, partially inhibited NK activation. Similarly, only anti-IFN alpha/beta antibodies partially blocked the proliferative response to loxoribine. In both cases, reconstitution of the responses was achieved by adding back IFN-alpha/beta to cultures containing antibodies against IFN-alpha/beta. These data suggest that, although several cytokines are produced in response to loxoribine, only IFN-alpha and IFN-beta are directly involved in the NK activation and proliferative responses. The pattern of strain variation appears to be reflective of variation in NK cell responsiveness to IFN-alpha/beta.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Citocinas/inmunología , Guanosina/análogos & derivados , Células Asesinas Naturales/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Ratones Endogámicos/inmunología , Animales , Línea Celular , Citocinas/biosíntesis , Pruebas Inmunológicas de Citotoxicidad , Ensayo de Inmunoadsorción Enzimática , Guanosina/farmacología , Humanos , Interferón-alfa/inmunología , Interferón beta/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Masculino , Ratones , Especificidad de la Especie , Bazo/citologíaRESUMEN
We have previously described a class of immunostimulatory ribonucleoside, exemplified by loxoribine (7-allyl-8-oxoguanosine) and 8-bromoguanosine, that act on cycling B cells to promote nonspecific proliferation and differention, and that synergize with Ag to recruit quiescent Ag-reactive B cells to undergo differentiation and high level Ab production. In murine B cells, two distinct binding activities have been characterized that have dissociation constants that parallel their distinctive dose-response profiles. The SJL mouse strain, which is hypo-responsive to the B cell proliferative properties of these molecules, exhibits a dissociation constant of 10- to 20-fold lower affinity than that of normal murine strains; its Kd for the higher affinity-binding interaction, however, is essentially normal. These observations suggested the existence of two distinct binding activities. Recently, immunoselective subgroups of loxoribine have been described, with an ability to stimulate individual components of the immune response that correlates with specific changes in discrete domains of the nucleoside molecule. By using a panel of nonstimulatory loxoribine analogues as inhibitors, we now report further evidence for the existence of unique sites on loxoribine binding proteins that recognize distinct domains on nucleoside analogues; changes in these regions confer immunologic selectivity. These data confirm the conclusions of previous studies, extend our understanding of the topology of subsites that are otherwise silent, and provide insight into the binding site mediating adjuvanticity. These results allow more profound appreciation of the nature of structural changes that will permit a tighter fit between ligand and binding site, ultimately promoting the design of more potent immunoselective agents.
Asunto(s)
Adyuvantes Inmunológicos/química , Linfocitos B/inmunología , Guanosina/análogos & derivados , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Animales , Sitios de Unión/inmunología , Células Cultivadas , Guanosina/inmunología , Técnica de Placa Hemolítica , Masculino , Ratones , Ratones Endogámicos CBA , Relación Estructura-ActividadRESUMEN
Leukemic B cells from a majority of patients with chronic lymphocytic leukemia (CLL) enter the cell cycle upon stimulation in vitro with loxoribine, a potent 7,8-disubstituted guanine ribonucleoside immunostimulant. In the absence of added costimulants, a proportion of these cells become activated and undergo DNA synthesis and mitosis accompanied by a marked increase in expression of an array of cell surface activation antigens. The resultant activated B-CLL cells exhibit greatly enhanced sensitivity to cycle-active cytotoxic drugs. This approach may be of potential value in the therapy of CLL.
Asunto(s)
Antineoplásicos/farmacología , Ciclo Celular/efectos de los fármacos , Guanosina/análogos & derivados , Leucemia de Células Pilosas/sangre , Leucemia Linfocítica Crónica de Células B/sangre , Linfocitos/efectos de los fármacos , Adulto , Anciano , Anticuerpos Monoclonales , Antígenos CD/sangre , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Guanosina/farmacología , Humanos , Inmunofenotipificación , Leucemia de Células Pilosas/inmunología , Leucemia de Células Pilosas/patología , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/patología , Activación de Linfocitos , Linfocitos/inmunología , Linfocitos/patología , Masculino , Persona de Mediana Edad , Mitosis/efectos de los fármacosRESUMEN
A series of 7,8-disubstituted guanosine derivatives was designed and prepared as potential B-cell-selective activators of the humoral immune response. These compounds were evaluated for their ability to act as B-cell mitogens and to augment the antibody response of B cells to sheep red blood cell (SRBC) challenge (adjuvanticity). In addition, they were tested for their ability to stimulate the natural killer (NK) cell response in murine in vitro cell assays. Certain of the compounds demonstrated in vivo activity when administered either intravenously, subcutaneously, or orally. Analogues with a medium-length alkyl chain (2-4 carbons, 5-7) on the 7-position of 7-alkyl-8-oxoguanosines were found to be particularly potent. Compounds bearing hydroxyalkyl, aminoalkyl, or substituted aminoalkyl substituents on this 7-position were weakly active. However, benzyl groups, including those substituted with heteroatoms (e.g., p-nitrobenzyl, 14), were active. Oxo, thioxo, and seleno groups on C-8 of the guanosine ring all imparted strong activity, whereas other larger substituents did not (e.g., N = CN). Stereochemical inversion of the 2'-hydroxyl on the ribose ring in this series, giving arabinose analogue 70, lessened activity. However, removal of the 2'-hydroxyl, either with (64) or without (73) removal of the 3'-hydroxyl, resulted in excellent activity and improved solubility; 64 also displayed good oral in vivo activity as well. A series of ketals involving the 2',3'-hydroxyls were prepared; certain of the nonpolar ketals (e.g., 48) were remarkably active, pointing to an ancillary hydrophobic binding region that can augment activity. 5'-Phosphate derivative 57 was fairly active, and acyclovir analogue 90 displayed good NK-selective activity: other N-9 sugar mimetics were also active (97-104), although this activity did not carry over into the human B-cell assay. A total of 80 compounds were prepared and evaluated for their immunostimulating activity. Within this group, compounds could be divided into those that were active in all three assays, those that displayed some measure of selectivity for the adjuvanticity assay, and those that preferentially activated NK responses. Because of its overall biological profile and ease of synthesis, 7-allyl-8-oxoguanosine (6; loxoribine, RWJ-21757) was chosen for further development. It is among the most potent compounds evaluated in the three biological assays.
Asunto(s)
Adyuvantes Inmunológicos/síntesis química , Guanosina/análogos & derivados , Animales , Linfocitos B/inmunología , Eritrocitos/inmunología , Guanosina/síntesis química , Guanosina/química , Guanosina/inmunología , Células Asesinas Naturales/inmunología , Linfoma/inmunología , Ratones , Ratones Endogámicos C3H , Mitógenos , Estructura Molecular , Ovinos/sangre , Bazo/inmunología , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
We have shown previously that loxoribine exhibits adjuvant activity for B cells, activates natural killer (NK) cells, and enhances the activation of lymphokine-activated killer cells by interleukin-2 (IL-2). In this study, we examined loxoribine for protective effects in a B16 melanoma lung tumor metastasis model. Significant inhibition of B16 metastasis was seen in mice given a single injection of 2 mg loxoribine as late as day 3 of tumor growth but the greatest inhibition (96%) was seen in mice given four injections of loxoribine on alternate days starting the day before tumor injection. In experiments in which both IL-2 and loxoribine were administered, both agents were active when tested alone, but the combination of IL-2 and loxoribine gave significantly greater inhibition of metastasis. Loxoribine partially inhibited the development of tumors in mice that had been depleted of NK cells by the administration of anti-asialo-GM1 or anti-NK1.1 antibodies and in NK-deficient beige mice. In all cases, protection was seen only when smaller tumor inocula were injected. Taken together, these data suggest that both NK and non-NK cell populations or effector mechanisms with antitumor activity were activated by loxoribine. Since substituted guanosine analogs have been shown to have adjuvant activity in B cell systems, we evaluated whether loxoribine was active as an adjuvant in a tumor protection model. Mice immunized with both irradiated tumor cells and loxoribine developed a significantly lower number of lung tumors when challenged by live B16 tumor cells, whereas mice injected with either vaccine or loxoribine alone were not protected. There was a clear dose response seen with both loxoribine and the vaccine preparations. These data suggest that loxoribine may be useful in tumor therapy as an immunomodulator or as an adjuvant for use with tumor vaccines.
Asunto(s)
Guanosina/análogos & derivados , Melanoma Experimental/tratamiento farmacológico , Adyuvantes Inmunológicos , Animales , Gangliósido G(M1)/inmunología , Guanosina/uso terapéutico , Interleucina-2/uso terapéutico , Neoplasias Pulmonares/secundario , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia/prevención & control , Vacunas/inmunologíaRESUMEN
7,8-Disubstituted guanine ribonucleosides represent a class of B lymphocyte agonists that utilize a protein kinase C-independent signaling pathway. These compounds provide an alternate T helper signal for B cells and enhance antigen-specific humoral responses in the murine model and in an IL-2-dependent human model in vitro. They effectively restore high level immune responses in a variety of murine models of immunodeficiency both in vivo and in vitro. In this study we examined the potential of these compounds to improve antibody responses generated by cultured cells from patients with common variable immunodeficiency (CVI). The inability to mount normal humoral responses to antigen was confirmed in nine patients with diagnosed CVI (CVI: 37 +/- 16, normal 653 +/- 116 plaque-forming cells (PFC)/culture; P less than 0.001). In cultured lymphocytes from eight of the nine patients studied, a normal level or greater responses to nominal antigen could be elicited by antigen in the presence of the immunostimulatory nucleoside 7-methyl-8-oxoguanosine (7m8oGuo). The average response to antigen increased from 37 +/- 16 without nucleoside to 1733 +/- 488 PFC/culture in its presence (P less than 0.002). Restoration of specific immune responses was an antigen-dependent and nucleoside dose-dependent event. Signaling by 7m8oGuo rendered the response to antigen protein kinase C independent in cultures of cells from normal donors as well as from CVI patients. These data substantiate (i) that a non-C-kinase signaling pathway for antigen-dependent differentiation exists, (ii) that this pathway can function normally in B cells from patients with CVI when triggered appropriately, and (iii) that 7,8-disubstituted guanine ribonucleosides can convert a C-kinase-dependent signaling event to a C-kinase-independent signaling event. Substituted guanine ribonucleosides may have potential as immunotherapeutic agents for patients with CVI.
Asunto(s)
Síndromes de Inmunodeficiencia/inmunología , Proteína Quinasa C/fisiología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Adulto , Alcaloides/farmacología , Formación de Anticuerpos/efectos de los fármacos , Antígenos CD/análisis , División Celular/efectos de los fármacos , Concanavalina A/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Guanosina/análogos & derivados , Guanosina/farmacología , Humanos , Inmunoglobulina A/análisis , Inmunoglobulina G/análisis , Inmunoglobulina M/análisis , Inmunofenotipificación , Técnicas In Vitro , Isoquinolinas/farmacología , Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Piperazinas/farmacología , Mitógenos de Phytolacca americana/farmacología , Inhibidores de Proteínas Quinasas , Transducción de Señal/fisiología , Esfingosina/farmacología , EstaurosporinaAsunto(s)
Adyuvantes Inmunológicos , Guanosina/análogos & derivados , Guanosina/farmacología , Animales , Formación de Anticuerpos/efectos de los fármacos , Guanosina/metabolismo , Humanos , Síndromes de Inmunodeficiencia/tratamiento farmacológico , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Linfocitos/metabolismoRESUMEN
7,8-Disubstituted guanine ribonucleosides are known to be potent intracellular modulators of immune responses. These compounds trigger and modulate a wide variety of lymphocyte responses including effects exerted directly on B cells. However, little is known about their mechanism of action. The current paper describes studies undertaken to evaluate whether binding components specific for these bioactive molecules exist in splenic B lymphocytes. After exposure of cells to labeled nucleoside, two different pools of nucleoside can be distinguished: a rapidly exchangeable nucleoside pool and a slowly exchangeable pool. The material in the latter pool consists of authentic unaltered nucleoside that is complexed to a relatively hydrophobic cellular component with an apparent Mr of 30,000-40,000; binding appears to interfere with free interaction of the nucleoside's cis hydroxyls with a boronate affinity resin. The slowly exchangeable nucleoside pool is seen to localize predominantly to the nucleus in electron microscopic autoradiographs. This pool is maximally bound by 30 min of incubation. Specific, saturable binding is demonstrable, with an apparent Kd of approximately 7 microM. This value correlates well with concentrations at which half-maximal biological activity occurs and suggests that the binding component likely mediates antigen-dependent immunomodulatory activity. Splenic B cells express approximately 2 x 10(4) binding sites/cell, whereas thymic lymphocytes, which do not respond functionally to nucleosides, do not display a measurable number of nucleoside binding sites. Ligand specificity of the binding interaction is confirmed by binding inhibition studies, in which binding inhibitory activity of unlabeled agonistic structural analogs recapitulate their degree of immunobiological activity. These data are most consistent with the existence of a saturable binding component with apparent specificity for 7,8-disubstituted guanine ribonucleosides in splenic B cells.
Asunto(s)
Linfocitos B/metabolismo , Guanosina/análogos & derivados , Animales , Autorradiografía , Linfocitos B/ultraestructura , Sitios de Unión , Células Cultivadas , Guanosina/metabolismo , Cinética , Masculino , Ratones , Ratones Endogámicos CBA , Microscopía Electrónica , Peso Molecular , TritioRESUMEN
C8-substituted guanine ribonucleosides activate B cells by a novel pathway that apparently is independent of GTP-binding proteins and protein kinase C. B lymphocytes from SJL mice are hyporesponsive to antigen-independent inductive signals transmitted by these nucleosides. In the current studies, the basis for this observation was explored. Responses of normal murine strains to these agents have been dissociated into antigen-independent (inductive) and antigen-dependent (differentiative) types by use of the 7,8-disubstituted guanine ribonucleosides. Dose-response profiles for inductive responses appear to correlate with apparent Kd values for low-affinity nucleoside binding sites; dose-response curves for antigen-dependent differentiative responses correlate with apparent Kd values for high-affinity binding sites. It was found that the SJL low-affinity site exhibits an apparent Kd that is approximately 10- to 20-fold lower in affinity for 8BrGuo than that of normal CBA mice. Although the low-affinity site in normal murine strains displays nearly equivalent affinity toward C8-substituted and 7,8-disubstituted nucleosides, the low-affinity site of SJL mice binds 7,8-disubstituted compounds with approximately 5-fold higher affinity than it does monosubstituted compounds. The dissociation constant for high-affinity nucleoside binding sites of SJL mice was only slightly different from that of CBA mice, consistent with the observation of essentially normal antigen-dependent nucleoside-mediated activity in SJL mice. The current observations support (a) a role for low-affinity binding sites in antigen-independent inductive events, (b) a role for high-affinity binding sites in antigen-dependent differentiative events mediated by substituted guanine nucleosides, and (c) the existence of aberrant low-affinity binding sites in B cells from SJL mice.
Asunto(s)
Linfocitos B/metabolismo , Guanosina/análogos & derivados , Síndromes de Inmunodeficiencia/metabolismo , Animales , Sitios de Unión/fisiología , Modelos Animales de Enfermedad , Guanosina/metabolismo , Cinética , Ratones , Ratones Endogámicos CBA , Ratones Endogámicos , Tionucleósidos/metabolismoRESUMEN
We have evaluated potential molecular mechanisms by which a group of synthetic lymphokine-like molecules, the 7,8-disubstituted guanine ribonucleosides, acts on second messenger pathways to augment the responses of murine B lymphocytes. Despite its extensive structural homology with GTP, 7-methyl-8-oxoguanosine (7-Me-8-oGuo), a prototypical disubstituted nucleoside, does not inhibit the binding of guanosine 5'-3-O-(thio)triphosphate either to purified G-proteins, or to G-proteins in situ in the plasma membrane. In contrast to anti-IgM antibodies, 7-Me-8-oGuo fails to induce elevation of intracellular free calcium in B lymphocytes. It does not elicit enhanced production of inositol phosphates in either unfractionated or large, cycling B cells (the cells on which it acts preferentially). It is unable to modify the cellular distribution or activity of protein kinase C, whereas phorbol 12-myristate 13-acetate, anti-IgD antibodies, and lipopolysaccharide do activate protein kinase C. Inhibitors of protein kinase C activity diminish stimulation of mRNA transcription by anti-IgD antibodies and lipopolysaccharide but not by 7-Me-8-oGuo. These data demonstrate that 7-Me-8-oGuo either uses a pathway distinct from that mediated by G-proteins, intracellular free calcium, inositol phosphates, and protein kinase C, or else bypasses the early events of this pathway, activating the cell at a point beyond their involvement. In either event, these nucleosides represent the only class of activator to date that is capable of driving B cell proliferation and differentiation without involvement of protein kinase C.
Asunto(s)
Linfocitos B/fisiología , Calcio/fisiología , Proteínas de Unión al GTP/fisiología , Guanosina/análogos & derivados , Activación de Linfocitos , Fosfatidilinositoles/fisiología , Proteína Quinasa C/fisiología , Animales , Guanosina/farmacología , Técnicas In Vitro , Ratones , ARN Mensajero/biosíntesis , Transducción de SeñalRESUMEN
The mechanism of action of a group of synthetic lymphokine-like molecules, the C8-substituted guanine ribonucleosides, was studied. Among their pleiotropic effects on B cells are the increased expression of surface Ia antigens, induction of polyclonal immunoglobulin secretion, enhancement of thymus-dependent as well as thymus-independent antibody responses, and transmission of T cell-like differentiative signals to B cells. However, relatively little is known about their molecular mechanism of action. In the current article, the interaction of 8-bromo-guanosine (8BrGuo), a prototypical C8-substituted guanine ribonucleoside, with cellular components was examined. Rapidly exchangeable (free) and slowly exchangeable (bound) 8BrGuo pools exist within B cells. The bound nucleoside pool loses its ability to be retained by a boronate affinity resin (despite its resistance to metabolic processing) and localizes to the cytosol on sucrose density gradients. Binding affinity, ligand specificity, and cellular specificity of binding all correlate closely with observed functional properties of these molecules. Together, these data suggest that the binding interaction mediates the biologic activities of 8BrGuo, and that the binding site acts as a functional nucleoside receptor.
Asunto(s)
Linfocitos B/fisiología , Animales , Sitios de Unión , Compartimento Celular , Diferenciación Celular , División Celular , Citoplasma/fisiología , Guanosina/farmacología , Cinética , Ligandos , Ratones , Ratones Endogámicos CBA , Bazo/metabolismoRESUMEN
As previously reported, LPS and 8-derivatized guanosine (both generators of IL-1 release), as well as IL-1 itself interfere with the in vivo induction of tolerance to DHGG in A/J mice. In the present studies it was demonstrated that desensitization of either A/J or CBA/CaJ mice with LPS aborts the ability of LPS to interfere with the induction of tolerance to DHGG. The abrogation of the ability of LPS to interfere with tolerance by LPS desensitization is not the result of neutralization of LPS by antibody produced to LPS during desensitization. Desensitization with LPS also aborts the interference with tolerance induction by 7-methyl-8-oxoguanosine. LPS desensitization inhibits the ability of LPS and/or 7-methyl-8-oxoguanosine to both convert a tolerogenic signal to an immunogenic signal and interfere with the induction of a tolerant state to a subsequent injection of Ag. The effects resulting from desensitization may be in part attributed to the depletion of IL-1. LPS desensitization also modulates the antibody response to injection of the AG, AHGG. Desensitization with LPS markedly suppresses the antibody response to a subsequent injection of AHGG in CBA/CaJ mice. Desensitization with LPS also inhibits the anti-HGG antibody response in A/J mice, but in this strain its effect is dependent on the route of injection of AHGG. In an experiment directly comparing the responses of normal and desensitized A/J mice to either intravenous or intraperitoneal injection of AHGG, desensitization only suppressed the response in mice injected with AHGG i.p.. Desensitization with LPS also inhibits the ability of LPS to act as an adjuvant in a subsequent antibody response to AHGG. Not only does desensitization interfere with the primary antibody response to AHGG, but it also interferes with the secondary response, suggesting that the primary injection after desensitization induces a state of immunologic tolerance.
Asunto(s)
Anticuerpos Antiidiotipos/biosíntesis , Desensibilización Inmunológica , Tolerancia Inmunológica , Interleucina-1/biosíntesis , Lipopolisacáridos/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Animales , Células Cultivadas , Inmunoglobulina G/administración & dosificación , Inmunosupresores/administración & dosificación , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Interleucina-1/fisiología , Ratones , Ratones Endogámicos A , Ratones Endogámicos CBA , Especificidad de la EspecieRESUMEN
Lymphokine-like activity and selective stimulation of B cell growth is exerted by a group of synthetic ribonucleosides derivatized at C8 and exemplified by 8-bromoguanosine (8BrGuo), 8-mercaptoguanosine, and 7-methyl 8-oxoguanosine. However, relatively little is known about their molecular mechanism of action. Like naturally occurring nucleosides, 8BrGuo is taken up into lymphocytes by a process of facilitated diffusion. Naturally occurring nucleosides are then reclaimed by a well characterized salvage pathway, involving sequential phosphorolysis and phosphoribosylation. The studies reported in this communication demonstrate that, in contrast to naturally occurring nucleosides, 8BrGuo is not a substrate for salvage by purine nucleoside phosphorylase. The base that would be produced by putative phosphorolysis, 8-bromoguanine, is biologically inactive and is not a substrate for hypoxanthine-guanine phosphoribosyl-transferase. Accordingly, inhibitors of purine nucleoside phosphorylase-mediated salvage fail to inhibit nucleoside-induced immunostimulation selectively. Examination of the metabolism of 8BrGuo provides no direct evidence that 8BrGuo is phosphorylated by B lymphocytes. Direct enzymatic phosphorylation does not seem to be essential to the mechanism of action of the nucleoside insofar as competitive inhibition of deoxycytidine kinase (an enzyme that directly phosphorylates purines as well as pyrimidines) or of deoxyguanosine kinase fails to inhibit 8BrGuo stimulation selectively. Moreover, studies with synthetic nucleosides in which 3' and/or 5' hydroxyl groups were irreversibly blocked, precluding their phosphorylation, demonstrated that immunobiologic activity can occur in the absence of 3' and/or 5' phosphorylation. Finally, experiments with radiolabeled nucleosides provide no evidence to support the hypothesis that they are incorporated into cellular nucleic acid. These data, together with previous studies, suggest that it is the unmetabolized nucleoside that is active and, as such, is most likely to act in a regulatory capacity.
Asunto(s)
Adyuvantes Inmunológicos/metabolismo , Guanosina/análogos & derivados , Adyuvantes Inmunológicos/farmacología , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , ADN/biosíntesis , Guanosina/metabolismo , Guanosina/farmacología , Humanos , Activación de Linfocitos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos CBA , Mitógenos/farmacología , Fosforilación , Inhibidores de la Síntesis de la Proteína/farmacología , ARN/biosíntesisRESUMEN
Production of an IL-1-like activity in cultures of irradiated splenic adherent cells can be elicited by the C8-substituted guanine ribonucleosides 8-bromoguanosine (8BrGuo) and 8-mercaptoguanosine (8MGuo). This report constitutes the first evidence for activation of a non-lymphocytic cell type by these agents to secrete an immunologically-active mediator. The secreted activity is mitogenic for murine thymocytes, co-stimulates these cells synergistically in the presence of concanavalin A, and co-stimulates low cell density cultures of purified B-cells in the presence of anti-mu antibodies. Production of this activity increases in a dose-dependent manner as the concentration of nucleoside is increased, both in cultures of splenic adherent cells and of the macrophage cell line P388D1. The P388D1 results indicate that this effect of the nucleoside is not mediated by another cell type, but can proceed by direct nucleoside-cell interaction. Optimal amounts of IL-1-like activity are produced after about 24 h of culture. Anti-IL-1 antibodies that neutralize the biologic activity of an IL-1 standard also eliminate the IL-1-like activity induced by 8BrGuo. These antibodies, however, fail to alter the magnitude of the primary humoral immune response to sheep erythrocytes amplified by 8BrGuo. These data indicate that C8-substituted guanosines, known intracellular stimuli for B-lymphocytes, can also induce non-lymphocytic cells (including a macrophage-like cell line) to elaborate an active principle which exhibits IL-1-like activity. These nucleosides thus are apparently able to elicit secretion of monokines, lymphokines and immunoglobulin from macrophages, T-cells and B-cells, respectively.