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Biomedical subjects

E Gronowicz

Publications and source records attributed to E Gronowicz.

At least 19 recordsLinked to original sources
PubMed1978

The polyclonal B cell activator dextran-sulphate induces formation of colony stimulating activity.

The effect of dextran-sulphate (DS), a polyclonal activator known to stimulate immature murine B-cells, was assayed in a culture system allowing the growth of myeloid cells. It was known that DS induced the production of a myeloid colony stimulating factor (CSF) by cells from both spleen and bone marrow. Nylonwool purified mouse spleen cells, enriched for T cells, showed a dimished CSF production in response to DS, while CSF production in response to Con A was increased. Furthermore, DS induced CSF in both spleen and bone marrow cells from nude mice. Removal of macrophages did not affect CSF production. The CSF induced was non-dialysable and no small molecular weight or lipoprotein inhibitors could be demonstrated. The results suggest that DS activates cells other than T cells or macrophages (possibly B cells or null cells) to produce a myeloid stem cell stimulating substance. These results indicate that interactions between lymphoid and myeloid cells can take place during differentiation.

Animals↗
PubMed1976

Spleen cells from animals tolerant to a thymus-dependent antigen can be activated by lipopolysaccharide to synthesize antibodies against the tolerogen.

Immunological tolerance was induced in adult mice by the injection of 5 mg of deaggregated hapten-protein conjugate. The tolerant state was confirmed 4-19 days later by the failure of such animals to mount an immune response against an aggregated form of the same thymus-dependent hapten-protein conjugate as well as by the inability of spleen cells from tolerant animals to respond to a thymus-independent hapten-carrier conjugate. Even though the animals were fully tolerant, their spleen cells were activated by lipopolysaccharide (LPS) in vitro to produce normal numbers of plaque-forming cells against the hapten. The finding that spleen cells from tolerant animals could be activated by LPS into synthesis of antibodies against the tolerogen indicates that tolerance to thymus-dependent antigens does not affect B cells, but presumably only T cells. It is suggested that the only stringent test for the existence of B-cell tolerance is the inability of polyclonal B-cell activators to activate antibody synthesis against the tolerogen. The findings make it unlikely that B-cell tolerance to autologous thymus-dependent antigens exists and further indicate that such antigens cannot deliver activating or tolerogenic signals to B cells, although they are competent to combine with and block the Ig receptors.

Animals↗
PubMed1976

Hapten-induced B cell paralysis. II. Evidence for trivial mechanisms of tolerance.

Injection into mice of a free reactive form of the hapten (4-hydroxy-3,5-dinitrophenyl)acetyl (NNP), induces a state of specific unresponsiveness to the hapten, upon its challenge with thymus-cependent and independent carriers. This unresponsiveness is maintained in vitro. Both induction and expression of the unresponsive state were found to be independent of T cells. Analysis of the mechanisms responsible for the B cell "tolerance" demonstrated that the major cause of unresponsiveness in this system is the blockade of specific surface receptors on B cells by the hapten, abolishing the focusing function of these receptors. "Tolerant" B cells, though they were unresponsive to the antigen, could be activated to anti-hapten antibody secretion by the B cell mitogen lipopolysaccharide (LPS), which does not require Ig-mediated binding. They also became fully responsive to the antigen NNP-LPS in specific thymus-independent responses after 24 hours of in vitro incubation in the absence of tolerogen, conditions which allow "deblocking" of Ig surface receptors, and, thus, restoration of the focusing function of these receptors. These results demonstrate that great care is required for a clearcut definition of B cell tolerance. In this example, the tolerant B cells were perfectly responsive to a competent ligand, and no indication of an altered triggering or effector processes was found. It appears that in this, as in many other cases of B cell tolerance, the systems or the animals are tolerant, whereas B cells maintain a resting nonactivated state and are fully responsive to the triggering signal. Thus, the existence of tolerance-inducing signals resulting in B cell unresponsiveness is questioned.

Antibody Formation↗
PubMed1976

A plaque assay for all cells secreting Ig of a given type or class.

A modification of the hemolytic plaque assay using protein A-coated red cells is described which makes use of the fact that the Fc portion of IgG binds to protein A. A number of murine plasmacytomas secreting different classes of Ig have been tested for plaque formation with these indicator red cells. In the presence of complement-binding antibodies specific for the corresponding class of secreted Ig, between 10 and 70% of all plated myeloma cells formed plaques. The assay shows a prozone effect in excess of antibody, suggesting that complexes of antibody and secreted Ig effect lysis of the target cells. This assay can be used to enumerate cells secreting any molecules for which complement-binding antibodies are available.

Bacterial Proteins↗
PubMed1976

Characterization of dextran-sulfate-sensitive cells.

The cell type participating in the mitotic response to the polyclonal B-cell activator (PBA) dextran-sulfate (DS) was investigated. Cells from murine fetal liver, adult bone marrow, and spleen were studied; only a limited number of all cells present in each organ responded to DS. Morphological studies of the activated cells showed the major population of activated cells in spleen to have the appearance of lymphoblasts. In bone marrow, several classes of hematopoietic cells were mitotically active, including mononuclear cells (lymphoblasts and monocytes), megakaryocytes, and myeloblasts. In these cultures, however, it was not possible to differentiate between DS-activated and spontaneously proliferating cells. Bone marrow and, to some extent, spleen cell cultures activated with DS contained a relative increase in numbers of phagocytic cells, whereas stimulation of spleen cells with lipopolysaccharide did not result in an increased phagocytosis. However, adherent cells were not necessary for activation of DNA synthesis by DS in spleen, and this cell type did not contribute to a measurable degree to the DNA synthetical response. DS cannot be regarded as a general stem cell mitogen for bone marrow cells since it failed to promote colony growth of hematopoietic cells in an in vitro system. However, supernatants from DS-activated spleen and bone marrow cell cultures did stimulate colony growth of murine bone marrow cells, indicating that stem cells of nonlymphoid origin might be indirectly activated by DS. In conclusion, the major cell population activated by DS in spleen is lymphocytes. In bone marrow, other cell types seem to participate in the response as well, but the activation mechanism may be indirect and not primarily the result of DS interaction with these cells.

Animals↗
PubMed1976

Heterogeneity of B cells: direct evidence of selective triggering of distinct subpopulations by polyclonal activators.

This investigation was undertaken to assess directly the previous postulate that distinct subsets of B cells in the adult mouse spleen are selectivity triggered by different polyclonal B-cell activators (PBA). Several strains of mice and lipopolysaccharide (LPS), purified protein derivative of tuberculin (PPD), and dextran sulphate (DS) were used in this study. Two experimental approaches were used: stimulation after addition of two PBAs simultaneously to cell cultures and eliminating the responding population to one PBA, by a hot pulse of radioactive thymidine, on a later response to another PBA. The results of these experiments indicated that DS stimulated a cell population completely different from that stimulated by the LPS- and PPD-sensitive cells. When LPS and PPD stimulations were compared, it was found that the cells responding to these PBAs were largely distinct, although some cells were sensitive to both these PBAs. The extension of the overlaps in these subsets (the number of cells that could be activated by either PBA) was found to vary from one strain to another. These experiments gave direct evidence of the existence of subsets that can be activated by different PBAs. The present results also provided indications of the functional performance of distinct subsets of B cells on activation.

Animals↗
PubMed1975

Genetical control of B-cell responses. IV. Inheritance of the unresponsiveness to lipopolysaccharides.

The inheritance of B-cell responsiveness to lipopolysaccharide (LPS) was studied in 55 crosses between mice of the low-responder strain C3H/HeJ and the high-responder strains B10.5M and C3H/Tif. F1 hybrid mice between the low-and the high-responder strains, showed in every case responses which were intermediate between the responses obtained with each parent. The responsiveness among F2 hybrid and backcross mice to either high- or low-responder parents, segregated into intermediate, high, or low categories, respectively. The present results are compatible with the hypothesis that responsiveness to LPS is determined by one single, codominantly expressed, autosomal gene. The capacity to develop a specific thymus-independent response to a hapten-LPS conjugate, also under genetical control, was found to segregate together with the capacity to develop polyclonal responses to LPS.

Animals↗
PubMed1975

Genetical control of B-cell responses. III. Requirement for functional mitogenicity of the antigen in thymus-independent specific responses.

Spleen cells from C3H/HeJ mice fail to respond with polyclonal antibody synthesis to mitogenic concentrations of lipopolysaccharide (LPS) which are optimal for activating spleen cells from a high-responder strain (B10.5M). This unresponsiveness is selective for LPS, since C3H/HeJ cells respond as normals to another B-cell mitogen, purified protein derivative of tuberculin. Spleen cells from low-responder mice also fail to mount a specific anti-NNP plaque-forming cell (PFC) response, when challenged in vitro by NNP-LPS. However, C3H/HeJ cells develop normal responses to another thymus-independent hapten conjugate, DNP-AECM-Ficoll. C3H/HeJ mice fail to mount a specific anti-LPS antibody response, when challenged in vivo with doses of soluble LPS which are fully immunogenic for the high-responder strain. However, C3H/HeJ mice develop normal direct and indirect PFC responses to LPS, when challenged with a thymus-dependent form of the immunogen. These results are interpreted as indicating as absolute requirement for functional mitogenicity of the antigen, in the induction of specific thymus-independent antibody responses.

Animals↗
PubMed1975

Mechanism of B-cell activation and paralysis by thymus-independent antigens. Additive effects between NNP-LPS and LPS in the specific response to the hapten.

Normal spleen cells showed a bell-shaped dose response profile when stimulated in vitro with the thymus-independent antigen (4-hydroxy-3,5-dinitrophenyl)acetyl (NNP)-lipopolysaccharide (LPS) with regard to the development of high-avidity plaque-forming cells to NNP. The addition of suboptimal concentrations of LPS to cultures stimulated by suboptimal concentrations of NNP-LPS resulted in optimal induction of B cells in that affinity fraction. Addition of LPS to cultures optimally stimulated by NNP-LPS resulted in paralysis of the specific cells. These results are interpreted in terms of the additive effects between the mitogenicity of LPS and the mitogenicity of NNP-LPS, the latter being selectively focused on the specific cells, thus providing further evidence for the 'one nonspecific signal' hypothesis for immune activation of B cells.

Adjuvants, Immunologic↗
PubMed1975

Genetic control of B-cell responses. I. Selective unresponsiveness to lipopolysaccharide.

Spleen cells from C3H/HeJ mice fail to develop both proliferative responses and increased polyclonal antibody secretion in the presence of concentrations of the B-cell mitogen lipopolysaccharide that are optimal for the induction of B-cell responses in conventional strains. This unresponsiveness is selective for lipopolysaccharide, since C3H/HeJ spleen cells respond normally to two other polyclonal B-cell activators-dextran-sulphate and purified protein derivative of tuberculin. These findings are interpreted as indicating a selective defect in the B-cell subpopulation that responds to lipopolysaccharides in conventional strains.

Activation Analysis↗
PubMed1975

Responsiveness of lymphoid precursors to polyclonal B-cell activators.

We have shown previously that in the differentiation of fetal liver cells to mature B cells in irradiated hosts, these cells sequentially gain responsiveness to the polyclonal B-cell activators dextran-sulphate (DxS), lipopolysaccharide (LPS), and purified protein derivative from tuberculin (PPD), in that order. In this paper we show that both fetal liver cells and adult bone marrow cells responded with proliferation to DxS, but not to LPS OR PPD. However, neither fetal liver nor bone marrow cells gave rise to detectable numbers of high-rate antibody-secreting cells on short-term stimulation by polyclonal B-cell activators. The lack of LPS and and PPD responses of fetal liver and bone marrow cells could not be ascribed to the presence of inhibitory cells, and the DxS-induced response in these cell populations was not dependent on adherent cells. However, LPS could inhibit the DxS response of fetal liver cells, possibly indicating that DxS-responsive cells are precursors to B cells. Direct evidence was provided that DxS activated B-cell precursors in bone marrow. Thus, this cell population became responsive to LPS after DxS prestimulation, as measured by DNA synthesis. Bone marrow cells, sequentially stimulated with DxS and LPS, contained increased numbers of cells with surface immunoglobulin, although no significant increase in numbers of antibody-secreting cells was obtained. These data indicate that DxS had the capacity to increase the rate of differentiation of B-cell precursors. Finally, we show that the sequential appearance of responsiveness in B-cell differentiation to polyclonal B-cell activators is not due to lack of accessory cells during early stages in maturation.

Animals↗
PubMed1975

Macrophages suppress direct B-cell activation by lipopolysaccharide.

The influence of macrophages on polyclonal B-cell responses to lipopolysaccharide (LPS) and on a primary, specific immune response to a hapten-LPS conjugate was studied in mouse spleen and lymph node cells in serum-free and serum-supplemented cultures. Macrophages were found not to be necessary, and B cells were directly activated by LPS. In serum-free cultures of macrophage-depleted spleen cells, (a) proliferation and antibody secretion occurred at normal levels or were enhanced when compared with normal spleen cells, (b) the responsiveness of limiting cell numbers to LPS was better than in normal spleen cell cultures, (c) LPS did not increase the number or activate residual macrophages, (d) the primary specific response to a hapten-LPS conjugate developed normally, (e) peripheral lymph node cells, which are known to contain a very low concentration of macrophages, from normal or congenitally athymic (nude) mice mounted excellent proliferative responses to LPS. Furthermore, in cultures supplemented with fetal bovine serum, depletion of adherent cells resulted in enhancement of LPS-induced B-cell responses. Addition of peritoneal macrophages to adherent cell-depleted spleen cells produced suppression at all concentrations of both mitogen and adherent cells. Suppressive activity could also be demonstrated in supernatants from adherent cell cultures stimulated by LPS.

Animals↗
PubMed1974

Mechanism of thymus-independent immunocyte triggering. Mitogenic activation of B cells results in specific immune responses.

The present experiments were performed in order to analyze the mechanism by which thymus-independent antigens (nonspecific B-cell mitogens) can induce specific immune responses to antigenic determinants present on the same molecule. The hapten NNP was coupled to the B-cell mitogen, lipopolysaccharide (LPS). The conjugate retained full mitogenic activity and bound specifically to NNP-reactive cells. NNP-LPS activated polyclonal as well as specific anti-NNP antibody synthesis, but the optimal concentrations for induction of specific anti-NNP cells were several orders of magnitude lower than the concentrations required for polyclonal activation. These low concentrations failed to activate nonspecific cells, but they induced specific thymus-independent responses of high-avidity NNP-specific cells with the typical kinetics of antigenic responses in vitro. Furthermore, hapten-specific cells were paralyzed by NNP-LPS concentrations that were optimal for induction of polyclonal activation. Specific activation and paralysis could be abolished by free hapten indicating that selective binding of NNP-LPS to hapten-specific cells was responsible for the specificity of the response. However, the triggering signal lacked specificity, since high-avidity specific anti-NNP cells could still be activated by stimulating concentrations of NNP-LPS in the presence of free hapten, even though the Ig receptor combining sites were presumably occupied by NNP. The findings show that B cells with specific Ig receptors for the antigenic determinants on mitogen molecules preferentially bind these molecules and become activated at concentrations still unsufficient to trigger other B cells that lack specific receptors. It is suggested that activation for primary IgM responses in B cells is the result of "one nonspecific signal." This nonspecific signal is provided by the mitogenic properties of some antigens (highly thymus independent or, alternatively, by nonspecific T-cell factors (for highly T cell-dependent antigens), or both, and the surface structures responsible for triggering are not the Ig receptors. The specific Ig receptors only act as passive focusing devices for nonspecific stimuli, entitling the cell to be selectively activated, even though both the signal and the receptors for the triggering are nonspecific.

Animals↗