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Biomedical subjects

E C Snow

Publications and source records attributed to E C Snow.

At least 37 records · Page 2Linked to original sources
PubMed1989

Phosphorylation of class I but not class II MHC molecules by membrane-localized protein kinase C.

Membranes were isolated from B cells stimulated with phorbol 12-myristate 13-acetate (PMA) for a time sufficient to allow maximal redistribution and activation of protein kinase C (PKC). Exposure of such membranes to a short incubation with [gamma-32P]ATP resulted in the detection of at least nine unique or hyperphosphorylated membrane proteins by SDS-PAGE and autoradiography. The appearance of these phosphoproteins was blocked by pretreatment of the membranes with H-7 or sangivamycin, two selective inhibitors of PKC. In addition, membranes purified from B cells treated with an inactive phorbol ester or stimulated with dibutyryl cAMP failed to exhibit a pattern of new phosphoproteins. These results are consistent with the involvement of PKC in the phosphorylation of the proteins. These phosphoproteins are also candidates for proteins whose functions are modified as a consequence of early signal delivery to resting B cells following membrane immunoglobulin occupancy. This system was utilized to identify the heavy chain of MHC class I molecules as one of the membrane proteins phosphorylated by PKC. The MHC class II molecules were not phosphorylated in membranes isolated from PMA-treated normal B cells or from PMA-treated B cells which had previously been exposed to IL-4. These results indicate that class I, but not class II, MHC molecules are phosphorylated by PKC. It is possible that such a modification of cell surface class I molecules may be involved during the process of signal transduction leading to B cell activation.

Animals↗
PubMed1988

Glycoprotein biosynthesis in B lymphocytes: induction of protein N-glycosylation, RNA synthesis, and DNA synthesis by phorbol ester plus ionomycin is blocked by protein kinase inhibitors.

The combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin produces a dramatic increase in the incorporation of [2-3H]mannose into Glc3Man9GlcNAc2-P-P-dolichol and glycoprotein, and the induction of RNA and DNA synthesis in murine splenic B lymphocytes (B cells). The kinetics of the induction processes and the concentrations of PMA and ionomycin required for the optimal response have been defined. While the levels of induction of RNA and DNA synthesis by PMA + ionomycin were similar to the mitogenic response to bacterial lipopolysaccharide, activation by PMA and the calcium ionophore resulted in a threefold higher stimulation in dolichol-linked oligosaccharide biosynthesis and protein N-glycosylation. These results indicate that all signalling mechanisms that trigger RNA and DNA synthesis may not be sufficient to produce maximal induction of the N-glycosylation apparatus. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent protein kinase C inhibitor, prevented the induction of protein N-glycosylation activity (IC50 = 11 microM), as well as RNA (IC50 = 18 microM) and DNA synthesis (IC50 = 12 microM), two common indices of B cell activation. N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) also inhibited the induction of oligosaccharide-lipid intermediate, glycoprotein, RNA, and DNA synthesis, but required higher concentrations than H-7 for 50% inhibition. N-(2-Guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a potent inhibitor of cyclic nucleotide-dependent protein kinases, had little effect on the activation of the B cell metabolic processes. The H-7-sensitive reactions involved in the induction of RNA and DNA synthesis occurred within 4 h, but induction of lipid intermediate and glycoprotein biosynthesis remained sensitive to H-7 for 10 h after exposure to PMA and ionomycin. Direct in vitro assays in the presence of 0.6% Brij 58 reveal that a cytosolic, phospholipid-dependent protein kinase activity is translocated to a membrane site(s) after treatment with PMA and ionomycin, and the translocated protein kinase is sensitive to H-7. The relative order of potency of the protein kinase inhibitors on the metabolic processes strongly supports the hypothesis that protein kinase C, acting synergistically with Ca2+ mobilization, plays a key regulatory role in the early stages of B cell activation. The synthesis of oligosaccharide-lipid intermediates and protein N-glycosylation are also shown to be induced in B cells activated by PMA + ionomycin.

Animals↗
PubMed1988

Induction of lymphokine responsiveness of hapten-specific B lymphocytes promoted through an antigen-mediated T helper lymphocyte interaction.

We describe a new experimental approach designed to detect signals transduced to B cells that have interacted, in an antigen-mediated mechanism, with helper T cells that cannot release soluble mediators. For this purpose, cells from an antigen-specific T helper cell line were treated with cyclosporin A (CSA). The stimulation of CSA-treated T cells with specific antigen in the presence of low concentrations of CSA, demonstrated that the T cells did not release detectable levels of interleukin-2, interleukin-4, and interleukin-5. When such CSA-treated T cells interacted with hapten-specific B cells in the presence of specific antigen, the B cells were found to develop responsiveness to exogenously added growth and differentiation inducing soluble mediators. The development of lymphokine responsiveness in such cultures could be partially blocked by the addition of a monoclonal antibody specific for major histocompatibility complex class II molecules expressed on the B cell surfaces. These results indicated that antigen-mediated interaction between B and T cells, in the absence of lymphokines, resulted in a phenotypic change in B cell behavior and suggested that the signal that promoted this change occurred as a consequence of the T cell antigen receptor binding to B cell surface Ia in association with processed antigen. This experimental system should afford an opportunity to determine the biochemical and molecular consequences in B cells that have interacted, by direct cell contact, with helper T cells.

Animals↗
PubMed1987

The phosphatidylinositol response is an early event in the physiologically relevant activation of antigen-specific B lymphocytes.

Receptor ligand-induced turnover of plasma membrane phosphatidylinositol (PI) has been implicated as part of a membrane receptor signal transduction system in a number of mammalian cell types. Signaling through B-lymphocyte surface immunoglobulin (sIg2) has been explored polyclonally through the use of anti-Ig reagents, with the assumption that anti-Ig mimics the process of antigen binding to the antigen-specific cell. We have utilized a method of obtaining trinitrophenyl (TNP)-specific populations of B lymphocytes in order to determine if antigen binding to these antigen-specific cells initiates PI turnover. This method has allowed us to explore the membrane phospholipid events following antigen binding directly, rather than with inference from the anti-Ig system. We have found that both thymus-dependent and thymus-independent antigens (with the exception of TNP-lipopolysaccharide) produced an increase in PI turnover comparable to that generated by anti-IgM stimulation. The lack of increased PI turnover following TNP-LPS stimulation may be attributable to the action of LPS on the biochemical events of the PI cycle. In a B-cell subpopulation depleted of antigen-specific cells, only anti-IgM produced a PI effect. These results represent the first demonstration of PI turnover as an early activation event in a physiologically relevant lymphocyte system.

Animals↗
PubMed1987

An evaluation of antigen-driven expansion and differentiation of hapten-specific B lymphocytes purified from aged mice.

Hapten-specific, trinitrophenyl antigen-binding B cells (TNP-ABC) were purified from inbred strains of mice representative of short-, intermediate-, and long-lived animals. Such populations of B cells were stimulated by either thymus-independent or thymus-dependent antigens in vitro and evaluated for both proliferation and differentiation into antibody-secreting cells. In the thymus-dependent system, the three strains of mice were selected on the basis of all being able to interact appropriately with the same T helper cell line. The results indicate that TNP-ABC purified from aged animals of all three strains responded to both forms of antigenic stimulation similar to TNP-ABC selected from young, littermate control animals. These results are discussed in terms of concepts of intrinsic B cell defects during the aging process.

Aging↗
PubMed1987

Induction of glycoprotein biosynthesis in activated B lymphocytes.

Resting murine splenic B lymphocytes (B cells) can be stimulated to proliferate by exposure to a variety of polyclonal activators. To investigate changes in glycoprotein synthesis that occur during the activation process, N-glycosylation activity was assessed by following the incorporation of [2-3H]mannose into dolichol-linked oligosaccharide intermediates and glycoprotein after B cells were exposed to anti-immunoglobulin M (anti-mu). Stimulation of B cells by anti-mu resulted in a dramatic induction of N-glycosylation activity. The incorporation of radiolabeled mannose into oligosaccharide-lipid increased 9-fold while the rate of labeling of glycoprotein increased 27-fold between 18 and 38 h after exposure to anti-mu. Maximal stimulation of N-glycosylation activity was observed at an anti-mu concentration of 20-50 micrograms/ml. Similar results were obtained when B cells were activated by bacterial lipopolysaccharide (LPS), another polyclonal activating agent. The major dolichol-bound oligosaccharide labeled during the induction period was determined to be Glc3Man9GlcNAc2 by HPLC analysis. Nearly full induction of oligosaccharide-lipid synthesis and protein N-glycosylation was also seen when DNA synthesis was suppressed by activating B cells with anti-mu in a serum-free medium, or by activating with anti-mu or LPS in the presence of hydroxyurea. The results suggest that the N-glycosylation pathway is induced during the G0 to G1 transition or during the G1 period, and that entry into S phase is not required. These studies describe a striking developmental increase in N-glycosylation activity and extend the information on biochemical changes occurring during the activation of B cells.

Animals↗
PubMed1986

Enhancement by monoclonal anti-Lyb-2 antibody of antigen-specific B lymphocyte expansion stimulated by TNP-Ficoll and T lymphocyte-derived factors.

By using antigen-specific populations of B cells (TNP-ABC) we have demonstrated that the type-2 antigen TNP-Ficoll was capable of initiating B cell proliferation only in the presence of T cell-derived factors. Monoclonal-anti-Lyb-2.1 antibody acted synergistically with a T cell-derived supernatant, as well as with B cell-stimulating factor (BSF-1) to enhance the level of B cell expansion obtained in this in vitro system. This effect of anti-Lyb-2.1 mAB was observed at each day of the antigen-driven B cell expansion and was seen only with B cells purified from strains expressing the Lyb-2.1 allele. The epitope density of hapten on the Ficoll plays a critical role in this process, because Ficoll that is haptenated with low density of hapten was not found to be stimulatory. These results suggest that the Lyb-2 surface molecule influences the antigen-driven B cell growth that is stimulated by type 2 antigens and BSF-1.

Animals↗
PubMed1986

Induction of the c-myc protooncogene after antigen binding to hapten-specific B cells.

The stimulation of hapten-specific B cell populations with the thymus-dependent antigen, TNP-KLH, was found to induce elevated levels of c-myc mRNA by 2 h. A similar treatment with carrier protein alone did not elevate c-myc mRNA above the level seen in the nonstimulated, resting B cells. These results indicate that antigen binding to the sIg receptor, in the absence of Th cell involvement, directly signals the antigen-binding cell and implicates the active participation of sIg during the process of antigen-mediated B cell activation.

Animals↗
PubMed1985

Characterization and function of autoreactive T-lymphocyte clones isolated from normal, unprimed mice.

Self-Ia-reactive cloned T-cell lines, designated PK, were established by long-term culture of T cells from normal DBA/2 mice with irradiated syngeneic splenic adherent cells (SAC), rich in macrophages and dendritic cells. The cell lines were Thy 1+, Lyt 1+, Lyt 2-, produced IL-2 following stimulation with syngeneic spleen cells, and did not exhibit alloreactivity when screened against six different H-2 haplotypes. Of the five cloned PK cell lines tested, four were I-Ed restricted while one was I-Ad restricted as determined by genetic mapping and blocking studies carried out with monoclonal anti-Ia sera. Extensive specificity studies suggested that the PK cells reacted to syngeneic Ia molecules alone and not to foreign antigens such as fetal calf serum (FCS) used in the culture medium, in association with self-Ia. SAC pulsed with FCS or other protein antigens such as turkey gamma-globulin (TGG) were tested for their ability to induce proliferation of autoreactive T cells and other antigen-specific T cells using culture conditions consisting of serumless medium and interleukin 2 (IL-2). The data showed that the autoreactive T cells proliferated better in response to antigen-unpulsed SAC, while FCS-specific and TGG-specific cell lines, developed independently, proliferated only in response to FCS- or TGG-pulsed SAC, respectively, but not to antigen-unpulsed SAC. These results clearly distinguished the autoreactive T-cell clones from the antigen-specific T-cell clones. Preliminary studies carried out to investigate the functions of autoreactive T cells suggested that these cells helped in the in vitro differentiation of alloantigen-specific cytotoxic T lymphocytes (CTL) from CTL precursors obtained from the thymus and augmented syngeneic, allogeneic, and antigen-specific immune responses in vitro. The autoreactive T cells were also capable of inducing both proliferation and differentiation of antigen-specific populations of B cells in the absence of antigen. The present investigation suggests that autoreactive, non-antigen-reactive T cells can be cloned from normal, unimmunized mice and that such cell lines may provide a powerful tool for analyzing the role of the syngeneic mixed lymphocyte reaction in induction and maintenance of both T-and B-cell immune responses.

Animals↗
PubMed1985

Preparation and analysis of antigen-specific memory B cells.

A procedure has been developed for the enrichment of TNP-binding memory B cells (TNP-MABC) from spleens of immunized mice. More than 75% of the cells expressed surface IgM (sIgM) and IgD (sIgD) and about 9% expressed surface IgG (sIgG). The TNP-MABC consisted of small resting lymphocytes with high affinity antigen-binding receptors. These cells expressed increased densities of Ia antigens and decreased densities of sIgD. Adoptive transfer of the cells into irradiated, carrier-primed syngeneic recipients resulted in their differentiation into IgG anti-TNP antibody-secreting cells. TNP-MABC secreted high affinity IgG anti-TNP antibodies when cultured in vitro with carrier-primed T cells and antigen. Limiting dilution analysis revealed that TNP-MABC contained a relatively low frequency of precursors for IgG-secreting cells that had an exceptionally large clone size. These results show that a highly enriched population of antigen-specific memory B cells can now be prepared and used to analyze their activation requirements.

Animals↗
PubMed1985

Insulin and growth hormone function as minor growth factors that potentiate lymphocyte activation.

The small resting lymphocyte is devoid of cell surface receptors for endocrine peptides such as insulin and growth hormone. During the process of activating these cells by either specific antigen or mitogenic agents the lymphocyte has been shown to display receptors for insulin. The present paper reviews work that indicates a functional role for both insulin and growth hormone during the process of lymphocyte activation. The results of these studies point to potentially important interactions between the immune and neuroendocrine systems and are discussed in terms of endocrine peptides playing a supportive role during the complex series of events that occur during lymphocyte activation.

Animals↗
PubMed1983

Activation of antigen-specific B cells: role of T cells, cytokines, and antigen in induction of growth and differentiation.

T cells and cytokines were used to activate highly enriched populations of 2,4,6-trinitrophenyl (TNP)-binding B cells (TNP-ABC). TNP-ABC did not proliferate or differentiate when they were cultured with thymus-dependent (TD) antigen, even in the presence of supernatants known to contain B-cell growth and differentiation factors. However, purified TNP-ABC did proliferate and differentiate when they were cultured with TD antigen in the presence of carrier-primed T cells and antigen (TNP-keyhole limpet hemocyanin)--i.e., linked recognition. TNP-ABC blasts generated under conditions of linked recognition proliferated and differentiated in response to cytokines in the absence of T cells and antigen. In contrast, under conditions of nonlinked recognition (hapten and carrier on different molecules) TNP-ABC blasts also proliferated but did not differentiate in response to the same cytokines. These results indicate that antigen-specific "resting" B cells must be activated by T cells and antigen prior to becoming responsive to cytokines. Furthermore, activation under conditions of linked and nonlinked recognition generates two different types of blasts with regard to their subsequent response to cytokines.

Animals↗
PubMed1983

Activation of antigen-enriched b cells. I. Purification and response to thymus-independent antigens.

The development of a procedure for the selection of a large number of functional antigen-binding B cells is described. Forty to 70% of the cells in the enriched population bind antigen, and the antigen-binding can be inhibited by pretreatment with either the free hapten (TNP-Lys) or anti-immunoglobulin. The enriched cells express both slgM and slgD and respond to mitogenic stimuli by proliferating and developing into antibody-forming cells. In contrast to normal spleen cells, however, the enriched cells also proliferate in response to submitogenic levels of TNP-Brucella abortus and TNP-LPS.

Animals↗
PubMed1983

Activation of antigen-enriched B cells. II. Role of linked recognition in B cell proliferation to thymus-dependent antigens.

The responsiveness to T-dependent (TD) and T-independent (TI) TNP-antigens of murine splenic B cells previously enriched for antigen-binding cells (ABC) was examined. TNP-TI antigens induced B cell proliferation. TNP-TD antigens did not induce a proliferative response regardless of the physical form or nature of the TNP-TD antigen (e.g., soluble vs particulate, low or high haptenation of carrier, TNP on various insoluble matrices, etc.). TNP-TD antigens were effective in enhancing the response of the TNP-ABC to all concentrations of lipopolysaccharide (LPS) tested, indicating that binding of antigen to surface immunoglobulin alters the LPS responsiveness of the cell. Irradiated, keyhole limpet hemocyanin- (KLH) primed T cells induced a threefold to fourfold greater B cell proliferative response with TNP-KLH than with fluoresceinated KLH (FLU-KLH) or FLU-KLH together with TNP-human serum albumin (TNP-HSA). Therefore, linked recognition appears essential for optimal T cell-mediated B cell proliferation, whereas the induction of B cell proliferation via nonlinked, carrier-activated T cells is a minor component of the response.

Animals↗
PubMed1981

The effect of growth hormone and insulin upon MLC responses and the generation of cytotoxic lymphocytes.

The ability of growth hormone (GH) and insulin to influence positively T lymphocytes responding to an alloantigen stimulus in vitro was analyzed through the use of a serum substitute system. The presence of insulin but not GH, enables the generation of a successful mixed lymphocyte culture (MLC) blastogenic response. However, the presence of GH during a 5-day MLC allowed for the generation of cytotoxic T lymphocytes (CTL). It was further demonstrated that GH needed to be present during the first 2 days of the culture system, presumably before the entry of the precursor CTL into cell division. The results are discussed in terms of the induction, by the GH, of ornithine decarboxylase activity and how this might relate to the successful generation of CTL activity.

Animals↗
PubMed1980

The role of insulin in the response of murine T lymphocytes to mitogenic stimulation in vitro.

The ability of insulin to influence the responsiveness of murine T lymphocytes in a culture system containing a serum substitute was documented. The presence of insulin was found to enhance the concanavalin A (Con A) reactivity of the lymphocytes. Once the cells were activated by a short-term exposure to Con A, insulin was capable of replacing Con A for the continued stimulation of the cells. This was true both for lymphocyte proliferation and for the generation of nonspecific cytotoxic T lymphocytes. The presence or absence of insulin was not found to influence the phytohemagglutinin responsiveness of the T lymphocytes. Possible reasons for the observed results are discussed in relation to a proposed model for lymphocyte activation.

Animals↗