RESUMEN
Gangliosides which are shed by tumor cells clearly inhibit cellular immune responses in vitro. However, the immunosuppressive activity of these molecules have been more difficult to ascertain in vivo. Here we have adapted a murine model to determine the effects of tumor gangliosides in an in vivo microenvironment, the lymph node draining the site of stimulation by allogeneic cells. In this model, allogeneic splenocytes (BALB/c) are s.c. injected into C3H mice. The cellular immune response in the draining popliteal lymph nodes 4 days later is evidenced as an increase in lymph node mass (2-fold), lymphocyte number (6-fold), and lymphocyte DNA synthesis (6-fold). Purified human neuroblastoma gangliosides (10 nmol) coinjected with the stimulating allogeneic cells significantly suppressed this in vivo immune response. The increase in the lymph node mass was reduced by 65% (0.66 versus 1.89 mg), the increase in lymphocyte number (4.0 x 10(6) cells/node) was almost completely inhibited (1.1 x 10(6) cells/node), and in vitro [3H]thymidine uptake by the lymphocytes recovered in vivo was reduced by 80%. In contrast to the inhibition by tumor gangliosides, liposomes of cholesterol:lecithin were not inhibitory. Thus, tumor gangliosides, specifically, modulate cellular immune responses in vivo, which may contribute to the observed enhancement of tumor formation by these molecules.
Asunto(s)
Gangliósidos/farmacología , Inmunidad Celular/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Proteínas de Neoplasias/farmacología , Bazo/citología , Animales , Gangliósidos/efectos adversos , Gangliósidos/metabolismo , Humanos , Liposomas/administración & dosificación , Liposomas/farmacología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Recuento de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Proteínas de Neoplasias/efectos adversos , Proteínas de Neoplasias/metabolismo , Neuroblastoma/metabolismo , Bazo/trasplanteRESUMEN
The injection of mice with a goat or rabbit antibody to mouse IgD stimulates a large polyclonal IgG response, approximately 10% of which is specific for antigenic determinants on the anti-IgD antibody molecule. The large goat IgG (GIgG)-specific antibody response in mice injected with goat antibody to mouse IgD requires that GIgG-specific B cells undergo much greater clonal expansion than B cells specific for other Ag. One possible explanation for the greater clonal expansion of GIgG-specific B cells is that B cells that lack GIgG specificity can only be stimulated with GIgG-specific T help during the relatively short time that anti-IgD binds to, and is processed and presented by, these B cells before they cease to express membrane mIgD. In contrast, GIgG-specific B cells can continue to bind, process, and present GIgG through mIgM after they lose mIgD. To test the hypothesis that extended stimulation with Ag-specific T help is required to generate a specific antibody response, we determined time requirements for Ag-specific T cell help for the development of such a response. Mice were injected with rabbit antibody to mouse IgD plus one or more daily injections of FITC conjugated to a F(ab')2 fragment of rabbit IgG (FITC-(Fab')2), which has a short in vivo half-life, and IgG1 anti-FITC antibody production was analyzed. In this system, each additional injection of FITC-F(ab')2 extends the period during which FITC-specific B cells can process this Ag and present it to rabbit IgG-specific T cells. Each additional injection of FITC-F(ab')2 stimulated a several-fold increase in IgG1 anti-FITC antibody levels, and injections on 5 consecutive days were required to induce a maximal anti-FITC response. These observations provide evidence that sustained Ag-specific T cell help is required to stimulate the degree of B cell clonal expansion that characterizes a specific antibody response.
Asunto(s)
Formación de Anticuerpos , Epítopos , Linfocitos T/inmunología , Animales , Anticuerpos Antiidiotipos/inmunología , Linfocitos B/inmunología , Femenino , Fluoresceína-5-Isotiocianato/inmunología , Haptenos/inmunología , Inmunoglobulina D/inmunología , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Cooperación Linfocítica , Ratones , Ratones Endogámicos BALB CRESUMEN
2-Amino-3-(methylamino)-propanoic acid (BMAA) is a neurotoxic, excitatory amino acid which has been linked through cycad use and consumption with the onset of a variant of amyotrophic lateral sclerosis occurring with high incidence in the western Pacific region. We have studied BMAA pharmacokinetics, oral bioavailability and blood-brain barrier permeability in the rat in an attempt to better define the possible role for BMAA in this disease. To evaluate its kinetics and uptake, BMAA (25-400 mg/kg) was administered to rats, either acutely or chronically, and then plasma and brain concentrations were determined at various times thereafter by combined gas chromatography mass spectrometry. After single dose i.v. injection, BMAA was cleared from plasma in a rapid distribution phase (Vd approximately 16 liters/kg) followed by a slower elimination phase (t1/2 approximately 1 day). Brain uptake was limited by a low blood-brain barrier permeability-surface area product of 2 to 5 x 10(-5) ml/sed/g. Brain BMAA levels peaked within 8 hr after injection, and then declined with a t1/2 similar to that of plasma. After two weeks of continuous infusion (100 mg/kg/day), steady-state brain concentrations equalled 10 to 30 micrograms/g, and only moderately exceeded those in plasma. The results suggest that BMAA may reach potentially toxic levels in brain (i.e., greater than 250 microM) after large doses (greater than 100 mg/kg). However, such doses are orders of magnitude greater than those available from dietary or medicinal use of cycads.
Asunto(s)
Aminoácidos Diaminos/farmacocinética , Barrera Hematoencefálica/efectos de los fármacos , Administración Oral , Aminoácidos Diaminos/sangre , Aminoácidos Diaminos/farmacología , Animales , Disponibilidad Biológica , Barrera Hematoencefálica/fisiología , Toxinas de Cianobacterias , Cromatografía de Gases y Espectrometría de Masas , Semivida , Inyecciones Intravenosas , Masculino , Ratas , Ratas Endogámicas , Distribución TisularRESUMEN
BSF-1, a cytokine produced by some T lymphocyte tumors, has been shown to act with anti-Ig antibodies to stimulate B lymphocyte proliferation, to independently induce resting B lymphocytes to increase their expression of surface Ia antigen, and to induce some activated B lymphocytes to differentiate into IgG1- or IgE-secreting cells. To determine whether BSF-1 might be secreted by normal lymphoid cells in the course of a physiologic immune response, BALB/c mice were injected with an affinity-purified goat antibody to mouse IgD (GaM delta), which induces the generation of a large, polyclonal T-dependent IgG1 response; 4-hr culture supernatants of spleen cells from these mice were prepared, and these supernatants were assayed for BSF-1 activity by analyzing their ability to induce BALB/c nu/nu spleen cells to increase their expression of cell surface Ia in vitro. Culture supernatants of unfractionated spleen cells removed from mice 4 to 8 days after GaM delta antibody injection induced substantial increases in B lymphocyte surface Ia expression; these increases were blocked by a monoclonal anti-BSF-1 antibody. Culture supernatants of spleen cells from untreated BALB/c mice or from untreated or GaM delta antibody-treated BALB/c nu/nu mice induced small to moderate increases in B cell surface Ia expression, and GaM delta antibody itself induced large increases in B cell surface Ia expression; however, these increases were not significantly blocked by a monoclonal anti-BSF-1 antibody. A culture supernatant of T cell-enriched spleen cells from untreated mice induced small increases in B cell surface Ia expression that were inhibited by anti-BSF-1 antibody, as was the larger increase in B cell Ia expression induced by a culture supernatant of T cell-enriched spleen cells from mice sacrificed 3 days after GaM delta injection. On the other hand, T cell-depleted spleen cells from BALB/c mice injected with GaM delta antibody 7 days before sacrifice failed to generate culture supernatants with BSF-1 activity. Supernatants prepared from spleen cells taken from untreated mice or mice treated with GaM delta antibody 1 to 3 days before sacrifice did not block the ability of purified BSF-1 to induce an increase in B cell surface Ia expression, and thus did not contain inhibitors of BSF-1 activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Linfocitos B/inmunología , Sustancias de Crecimiento/biosíntesis , Linfocinas/biosíntesis , Linfocitos T/inmunología , Animales , Anticuerpos Antiidiotipos/inmunología , Células Cultivadas , Medios de Cultivo , Sustancias de Crecimiento/metabolismo , Antígenos de Histocompatibilidad Clase II/análisis , Inmunoglobulina D/inmunología , Interleucina-4 , Linfocinas/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos/inmunología , Bazo/inmunología , Factores de TiempoRESUMEN
The injection of mice with 800 micrograms of an affinity-purified goat antibody to mouse IgD (GaM delta) induces early, T-independent polyclonal increases in the expression of B cell surface Ia, and B cell size and DNA synthesis, as well as later, T-dependent polyclonal increases in spleen cell number and Ig secretion. We have now studied the effects of varying the doses of injected GaM delta on all phases of B cell activation, as well as the effects of supplementing GaM delta with varying quantities of normal goat IgG (GIgG). We have found that while 12.5 micrograms of GaM delta modulates most of the IgD from the surface of splenic B lymphocytes, it fails to activate these cells. Increases in the expression of B cell surface Ia are first seen when 50 micrograms of GaM delta is injected, while increases in B cell DNA synthesis usually require the injection of 200 micrograms of GaM delta and peak with doses of approximately 800 micrograms. Increases in splenic B cell number and DNA synthesis during the T-dependent phase of GaM delta-induced B cell activation are seen only in those mice that were injected with sufficient quantities of GaM delta to induce DNA synthesis during the T-independent phase. Supplementing the dose of GaM delta injected with additional GIgG has no significant effect on B cell DNA synthesis or B cell number but dramatically increases polyclonal IgG1 secretion. Although mice which have been injected with 50 micrograms of GaM delta or with 800 micrograms of GIgG alone have few polyclonal IgG1-secreting cells, substantial increases in the number of IgG1-secreting cells are seen in mice injected with 50 micrograms of GaM delta plus 750 micrograms of GIgG. GIgG and larger doses of GaM delta similarly act synergistically to increase polyclonal IgG1 secretion. In contrast to the induction of polyclonal IgG1 secretion, the stimulation of polyclonal IgM secretion requires the injection of mitogenic doses of GaM delta and is not enhanced by the injection of additional GIgG. These observations suggest that, in this model system, stimulatory signals that activate B cells through their surface Ig are limiting for the induction of polyclonal proliferation and IgM secretion, while the generation of T helper lymphokines that do not directly interact with B cells through their surface Ig may be more limiting for the stimulation of polyclonal IgG1 secretion.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Linfocitos B/inmunología , Inmunoglobulina D/inmunología , Activación de Linfocitos , Animales , Diferenciación Celular , Cabras , Cadenas delta de Inmunoglobulina/inmunología , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Previous studies from this laboratory have shown that the injection of mice with an affinity-purified goat antibody to mouse IgD (GaM delta) induces T-independent polyclonal increases in: 1) B cell DNA synthesis, and 2) expression of surface Ia antigen and receptors for T helper factors, 1 to 2 days after injection. In addition, T-independent polyclonal increases in B cell number and IgG1 secretion are observed 6 to 7 days after injection. The administration of normal goat IgG (GIgG) along with GaM delta has been shown to augment GaM delta-induced polyclonal IgG1 secretion. To obtain information about the characteristics of the T help that is required for polyclonal antibody production in this model system, we investigated: 1) the length of the period during which GaM delta must be present to induce day 7 polyclonal antibody production, and 2) the kinetics of the induction of splenic T cell DNA synthesis. We found that GaM delta can be neutralized 3 days after injection by the administration of IgD without decreasing day 7 polyclonal IgG1 secretion, as long as mice are given GIgG at the time that GaM delta is neutralized. In contrast, polyclonal IgG1 secretion is greatly inhibited if GaM delta is neutralized 1 to 2 days after injection or if GaM delta is neutralized 3 days after injection, but GIgG is not administered at this time. Because GIgG can stimulate activated GIgG-specific T cells to secrete helper factors, but, unlike GaM delta cannot focus GIgG-specific T help polyclonally onto B cells, these findings suggest that nonspecific T help, rather than antigen-specific T help, is required in this system after day 3 for the induction of polyclonal IgG1 secretion. Determination of the kinetics of the induction of T cell DNA synthesis in this system by in vivo [3H] thymidine incorporation studies, as well as dual laser fluorescence-activated cell sorter analysis of T and B cell DNA content, indicate that T cells are induced to synthesize DNA 2 days after GaM delta injection and reach plateau rates of DNA synthesis 3 days after injection. Taken together, the GaM delta neutralization experiments and DNA synthesis studies suggest that one reason that GaM delta is required for 3 days in this system is to allow maximal activation of GIgG-specific T cells, which when stimulated later by GIgG secrete nonantigen-specific helper factors that induce GaM delta-activated B cells to secrete IgG1.