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S K Dower

Publications and source records attributed to S K Dower.

At least 109 records · Page 6Linked to original sources
PubMed1988

Abnormal intracellular sorting of O-linked carbohydrate-deficient interleukin-2 receptors.

The synthesis and intracellular sorting of the interleukin-2 (IL-2) receptor were studied with a line of mutant Chinese hamster ovary (CHO) cells with a reversible defect in protein O glycosylation. Under normal culture conditions the mutant ldlD cannot add N-acetylgalactosamine (Ga1NAc) to proteins. Ga1NAc is the first sugar of mucin-type O-linked oligosaccharides attached to protein. This O-glycosylation defect is rapidly corrected when Ga1NAc is added to the culture mediu. An expression vector for the p55 human IL-2 receptor was transfected into wild-type CHO and ldlD cells and the structure, stability, and cell surface expression of the receptor were examined by immunoprecipitation and antibody-binding assays. Essentially all of the mature form of the normally glycosylated IL-2 receptor in both wild-type CHO cells and ldlD cells incubated with Ga1NAc was expressed on the cell surface. The stability of O-linked carbohydrate-deficient (Od) IL-2 receptors (in ldlD cells without Ga1NAc) was normal; however, missorting of the Od receptors resulted in very little cell surface expression. The sialidase sensitivity and endoglycosidase H resistance of mature Od IL-2 receptors suggest that Od receptor missorting occurred in or beyond the trans Golgi apparatus. The abnormal sorting of the Od IL-2 receptor is compared with the O-glycosylation dependence of the surface expression and stability of the low-density lipoprotein receptor, decay-accelerating factor, and the major antigen envelope protein of Epstein-Barr virus.

Animals↗
PubMed1988

Development and characterization of two neutralizing monoclonal antibodies to human interleukin-1 alpha.

Mice immunized with homogeneous recombinant interleukin-1 alpha (IL-1 alpha) protein developed specific serum titers to the immunogen. Hybridomas resulting from the fusion of the immune spleen or lymph node cells to myeloma cells were analyzed by an antibody capture assay in which the antigen was present in solution. This assay enabled us to isolate two hybridomas secreting antibodies (designated 2F4 and 4G12) that recognized IL-1 alpha and not interleukin-1 beta as judged by the ability of the antibodies to: (a) precipitate IL-1 alpha, (b) inhibit the binding of 125I-IL-1 alpha to the IL-1 receptor on EL4 cells, (c) inhibit the biological activity of IL-1 alpha as measured in a lectin-induced, IL-1-dependent thymocyte proliferation assay. In a double determinant assay configuration, both antibodies, in conjunction with rabbit polyclonal anti-IL-1 alpha antibodies, could detect nanogram concentrations of IL-1 alpha in solution. Cross-inhibition studies indicated that the 2F4 and 4G12 antibodies bind to the same or spatially related epitopes since each can inhibit the binding of the other to IL-1 alpha.

Animals↗
PubMed1987

Targeting of murine radiolabeled monoclonal antibodies in the lymphatics.

The tissue localization of a radiolabeled monoclonal antibody directed against a mouse Class I major histocompatibility antigen has been determined in mice following i.v. and s.c. administration. When labeled antibody was given s.c., radioactivity rapidly accumulated in regional lymph nodes draining the injection site, allowing visualization of the nodes by gamma camera imaging within minutes of injection. At 2 h after s.c. injection, radioactivity in regional nodes was present largely as intact antibody, but considerable degradation of antibody present in nodes was noted by 12 h after injection. Since little of the radioactivity reached the blood stream, visualization of regional nodes was possible for long periods after dosing. In contrast, antibody given i.v. showed no significant accumulation in lymph nodes at any time after dosing.

Animals↗
PubMed1987

The interleukin-1 receptor binds the human interleukin-1 alpha precursor but not the interleukin-1 beta precursor.

Both IL-1 alpha and IL-1 beta are initially translated as approximately Mr 30,000 polypeptides and processed to approximately Mr 17,500 prior to, or during, release from macrophages. The current study utilizes an in vitro transcription-translation system to produce these four forms of IL-1 directly from cloned cDNAs, in order to investigate the relative receptor binding and biological activities of the proteins. The data show that the initial translation product from IL-1 beta mRNA must be processed in order to bind to the IL-1 receptor and hence express biological activity. By contrast, the initial translation product of IL-1 alpha mRNA can bind to the IL-1 receptor without further proteolytic processing. These data suggest that the IL-1 biological activity previously associated with proteins having Mr values in the range 30,000-40,000 is due to IL-1 alpha gene products.

Binding, Competitive↗
PubMed1987

Determination of the minimum polypeptide lengths of the functionally active sites of human interleukins 1 alpha and 1 beta.

Interleukin 1 (IL-1) is a two-member family of proteins (IL-1 alpha and IL-1 beta) that mediates a diverse series of immune and inflammatory responses. These two proteins have only 26% amino acid homology yet bind to the same receptor. It is of importance to define the active sites of these molecules in order to understand their receptor interactions and the mechanisms involved in their multiple biological functions. We report here the localization of the biologically active portions within the initial polypeptide translation products. An in vitro transcription and translation system was used to generate specific fragments of each of the IL-1 molecules, which then were assayed for receptor binding capability and biological activity. Using this system, we have demonstrated that core sequences of amino acids for IL-1 beta (numbers 120-266) and 140 amino acids for IL-1 alpha (numbers 128-267) must be left intact to retain full biological activity and further that the biological activities of the IL-1 polypeptides parallel their receptor binding capabilities.

Amino Acid Sequence↗
PubMed1986

High level stable expression of human interleukin-2 receptors in mouse cells generates only low affinity interleukin-2 binding sites.

A bovine papilloma virus-derived vector was used to direct the high level expression in mouse C127 cells of three different cDNAs encoding the human interleukin-2 receptor. These were: the previously described cDNA clone isolated from the T-cell lymphoma, HUT-102; a cDNA clone isolated from mitogen-activated, normal peripheral blood T cells; and an altered version of the HUT-102 receptor in which Ser247, believed to be the site of protein kinase C-mediated phosphorylation, has been changed to an Ala residue. Fluorescence-activated cell-sorting using a monoclonal antibody directed against the human IL-2 receptor was used to derive stable lines of C127 cells expressing from 2-6 X 10(6) IL-2 binding sites per cell. However, all of these receptors bound IL-2 with low affinity.

Amino Acid Sequence↗
PubMed1986

Similarity between the interleukin 1 receptors on a murine T-lymphoma cell line and on a murine fibroblast cell line.

Interleukin 1 beta (IL-1 beta), one of two different polypeptide hormones with interleukin 1 (IL-1) biological activity, produced by activated human monocytes, is a 17.5-kDa protein. IL-1 beta binds specifically to a variety of cells; the cellular distribution of binding is consistent with reported biological responsiveness. In this report we show that two unrelated, but IL-1-responsive, cell lines, LBRM-33-1A5, a T-lymphoma line, and BALB/3T3, a fibroblast line, bind 125I-labeled IL-1 beta via similar plasma membrane receptor molecules. The T-lymphoma cells possess 238 +/- 16 plasma membrane receptors per cell and bind 125I-labeled IL-1 beta with an affinity of 3.6 +/- 0.9 X 10(9) M-1. The IL-1 receptor has a molecular size of approximately equal to 79.5 kDa, as estimated by affinity cross-linking. The fibroblasts possess 4.8 +/- 0.5 X 10(3) IL-1 receptors per cell and bind 125I-labeled IL-1 beta with an affinity of 2.6 +/- 0.5 X 10(9) M-1. The molecular size of the receptor molecule on the fibroblasts is approximately equal to 78 kDa. Despite the similarity in the characteristics of the ligand-receptor system on the two different cell types, the biological responses of the two cell types to IL-1 beta occur at IL-1 beta concentrations that differ by four orders of magnitude.

Animals↗
PubMed1986

Decreased production of interferon-gamma by human neonatal cells. Intrinsic and regulatory deficiencies.

Human neonatal lymphocytes produced little macrophage activation factor in response to mitogens. This correlated with decreased production of interferon-gamma (IFN gamma): adult lymphokines contained 894.2 +/- 177.1 U/ml, whereas neonatal cord and peripheral lymphokines contained 66.9 +/- 17.0 and 116.7 +/- 29.6 U/ml by bioassay. Results by radioimmunoassay (RIA) for IFN gamma were similar. In contrast, the interleukin 2 content of cord lymphokines was greater (P less than 0.01) and that of neonatal peripheral blood lymphokines similar to that of adults. Interleukin 1 production and interleukin 2 receptor expression and affinity were similar for adult and neonatal cells. Interleukins 1 and 2 in amounts comparable to those in adult lymphokines did not increase production of macrophage activation factor or IFN gamma by neonatal cells. Neonatal cells did not contain intracellular IFN or degrade exogenous IFN. Excess suppressor activity was not found in neonatal cultures. Addition of IFN alpha, 10,000-50,000 U/ml of interleukin 2 or phorbol myristate acetate (PMA) to cord mononuclear cells or of adult monocytes or PMA to cord T cells increased IFN gamma production compared to cells stimulated with concanavalin A (ConA) alone. Nevertheless, under optimal conditions (T cells + PMA + Con A), adult cells produced much more IFN gamma (1,360 +/- 261 U/ml by RIA) than cord cells (122 +/- 37 U/ml). Staphylococcal enterotoxin A (SEA) stimulated cord cell IFN gamma production at low cell densities; nevertheless, adult cells produced more IFN in response to SEA 1,341 +/- 350 U/ml) than cord cells (350 +/- 33 U/ml). Decreased production of IFN gamma by neonatal cells appears to be due both to differences in their intrinsic capacity to produce IFN gamma and to differences in regulatory mechanisms.

Concanavalin A↗
PubMed1985

Detection and characterization of high affinity plasma membrane receptors for human interleukin 1.

Interleukin 1 (IL-1) is a polypeptide hormone that acts as a central mediator of inflammation. Since IL-1 action is presumably mediated by specific cell surface receptor(s), we have characterized the binding of this hormone to cells. Purified human IL-1 was labeled to high specific activity with 125I, using Bolton-Hunter reagent. The labeled protein binds specifically to LBRM-33-1A5 (a murine T lymphoma line previously shown to produce IL-2 in response to phytohemagglutinin and IL-1) with an affinity of approximately 0.2-2 X 10(10)/M and, at saturation, to approximately 500 receptors per cell, on intact cells at 8 degrees C in the presence of sodium azide. The affinity of unmodified IL-1 for the murine plasma membrane receptor is 0.9-2 X 10(10)/M, as measured by the inhibition of 125I-IL-1 binding. The murine receptor specificity has been confirmed by demonstrating that, among a series of 12 polypeptide hormones, only IL-1 inhibits 125I-IL-1 binding to LBRM-33-1A5 cells. Treatment of surface-bound 125I-IL-1 with bivalent water-soluble crosslinkers identified a membrane polypeptide of Mr 79,500 to which IL-1 is crosslinked. A variety of cell types have been surveyed for the capacity to bind 125I-IL-1 specifically. The presence of specific binding correlates with the capacity of the cells tested to respond to IL-1. Our results indicate that the biological effects of the polypeptide hormone IL-1 are mediated by high affinity plasma membrane receptors. The identification of these receptors should provide valuable insight into the apparently diverse biological activities of IL-1.

Animals↗
PubMed1985

Quantitative measurement of human interleukin 2 receptor levels with intact and detergent-solubilized human T-cells.

2A3 monoclonal antibody (gamma 1, kappa) is a novel high-affinity reagent for detecting the human interleukin 2 (IL-2) receptor. The antibody inhibits IL-2 binding to its receptor and is an antagonist of IL-2 action. Detailed analysis of the mechanism of binding of the IgG, (Fab')2 and Fab' of 2A3 antibody shows that the bivalent species cross-link on the cell surface when bound. Measurements of IL-2 receptor expression on digitonin-permeabilized cells suggest that the intracellular pool of receptors is small. The antibody will bind to IL-2 receptors on glutaraldehyde-fixed cells in the presence of Triton X-100. This property is used in designing an assay for quantitative measurements of IL-2 receptor concn in solution. This assay can be used to monitor receptor protein during purification to homogeneity.

Animals↗
PubMed1985

Interaction of monoclonal antibodies with MHC class I antigens on mouse spleen cells. II. Levels of expression of H-2K, H-2D, and H-2L in different mouse strains.

The numbers of MHC class I molecules expressed by spleen cells from various mouse strains were determined by using MHC-specific monoclonal antibodies and a radioactive binding assay. Although small differences were found to exist in some cases, our general conclusion is that different mice of the same strain, congenic mice of different haplotypes, and syngeneic mice of varying background all express similar numbers of class I antigens. B10.A mice (8 to 10 wk old), for example, express 5.3 X 10(4) Kk molecules/cell, 5.4 X 10(4) Dd molecules/cell, and 2.2 X 10(4) Ld molecules/cell. Some of the differences observed in class I antigen expression included: 1) the level of Kk expression increased to a small but significant extent with age in B10.A mice; 2) female B10.A mice expressed slightly higher amounts of Kk than male mice; and 3) B10.A(2R) and B10.A(4R) recombinant strains expressed elevated levels of K-end antigens and slightly decreased levels of D-end antigens when compared with the unrecombinant B10.A strain. In several strains, F1 mice express approximately 50% as many copies of each parental antigen as do the homozygous parents. B10 mice, which are negative for the L antigen, nevertheless express the same total number of D-end molecules as do B10.A mice. The data suggest that the levels of expression of MHC class I molecules are controlled by at least two factors: gene dosage and another factor(s) that gives rise to the small variations in class I antigen expression seen with age, sex, and strain, and to the low expression of Ld relative to Dd and Kk.

Animals↗
PubMed1985

Induction of antibody responses to influenza virus in human lymphocyte cultures. I. Role of interleukin 2.

The in vitro T cell-dependent antibody response of human lymphocytes to influenza virus X31 was used to study the role of T cell-derived lymphokines in antigen-specific responses. Supernatant from cultures of phytohaemagglutinin-stimulated, pooled human tonsil cells (PHA-MLR) was capable of replacing T cells and inducing T-depleted tonsil cells to secrete influenza-specific antibody. The T cell-replacing activity of PHA-MLR supernatant co-purified with interleukin 2 (IL 2) on Ultrogel AcA54 gel filtration and reversed phase-high performance liquid chromatography. PHA-MLR supernatant and IL 2 also enhanced B cell proliferation induced by anti-mu or Staphylococcal aureus strain Cowan I (SAC). A murine monoclonal antibody directed against the human IL 2 receptor (Mab 2A3) was used to completely block the enhancement of influenza-specific antibody production mediated by PHA-MLR supernatant, purified IL 2, and recombinant human IL 2. Mab 2A3 did not affect the T-independent B cell proliferation induced by anti-mu or SAC, but abrogated the enhancing effect of the PHA-MLR supernatant and IL 2 in this culture system. Immunofluorescence studies failed to demonstrate binding of Mab 2A3 to B cells activated by the X31 influenza virus and IL 2, or by SAC. By using Mab 2A3 to mask out IL 2 effects in the influenza-specific culture system, no other B cell differentiating activities were revealed in supernatants from lymphocytic cultures stimulated with a variety of mitogens. Thus, our results indicate that the production of influenza-specific antibodies by T-depleted human lymphocyte cultures is absolutely dependent on the presence of both antigen and IL 2.

Antibodies, Monoclonal↗
PubMed1984

Trinitrophenyl modification of H-2k and H-2b spleen cells results in enhanced serological detection of Kk-like determinants.

Several anti-H-2Kk but not anti-H-2Dd monoclonal antibodies (mAb) exhibited enhanced binding to B10.A murine spleen cells after modification of the cells with trinitrobenzene sulfonate (TNBS). The number of antibody molecules bound to TNP-modified B10.A spleen cells increased by a factor of two or more. The same anti-2Kk mAb that exhibited enhanced binding to modified B10.A cells did not bind to unmodified C57BL/10 spleen cells, as expected, but did bind to TNP-modified C57BL/10 spleen cells. This TNP-dependent binding was not a result of cross-reactions with cell surface TNP groups nor with Fc receptors. TNP modification of a variant cell line that does not express class I H-2 products did not result in enhanced binding by these mAb. These findings can account for preferential recognition of TNP-Kk by B10.A and B10.BR CTL, and also for cross-reactive lysis by C57BL/10 CTL stimulated by C57BL/10-TNP against unmodified H-2Kk targets.

Animals↗
PubMed1984

Purification and chemical characterization of the receptor for interleukin 2 from activated human T lymphocytes and from a human T-cell lymphoma cell line.

The cell surface receptor for interleukin 2 plays a central role in the biology of this T-cell growth factor. A combination of affinity chromatography, high-performance liquid chromatography, and NH2-terminal protein sequencing was used to purify and chemically characterize the interleukin 2 receptor both from phytohemagglutinin-activated T cells and from the human T-cell lymphoma cell line HuT-102. The receptor isolated from HuT-102 cells was purified 16,000-fold to homogeneity as evidenced by (i) a final specific activity close to the theoretical specific activity of 18,182 fmol of receptor per microgram of protein, (ii) a single band on polyacrylamide gel electrophoresis with an Mr of 55,000, and (iii) a unique, unambiguous NH2-terminal protein sequence. The receptor purified from phytohemagglutinin-activated T lymphocytes had an Mr of 60,000 but it had the same NH2-terminal protein sequence.

Amino Acid Sequence↗
PubMed1984

Limiting dilution analysis of interleukin 2 and colony-stimulating factor producer cells in normal and autoimmune mice.

MRL/MP lpr-lpr (MRL-lpr) mice spontaneously develop an age-related disease characteristic of human systemic lupus erythematosus (SLE). Old MRL-lpr mice (4 mo of age) develop antibodies to nucleic acids, display immune complex glomerulonephritis, and have a massive T cell-associated lymphadenopathy. In concert with disease development is data showing an age-related loss of interleukin 2 (IL 2) production by mitogen-stimulated lymphoid cells from these mice. The loss of IL 2 production has been suggested to be involved in the onset and/or development of autoimmune disease seen in these animals. In this report, we examined the frequency of both IL 2 and colony-stimulating factor (CSF) producer T cells in the MRL-lpr mouse by using a limiting dilution analysis assay. Our results show that the number of IL 2 and CSF producer cells present in autoimmune animals is similar to the number found in normal control mice. In addition, IL 2 and CSF producer T cells from autoimmune MRL-lpr mice make similar levels of lymphokine activity, as do producer T cells from normal mice. Our data argue against the previously hypothesized role that a paucity of IL 2 production may be involved in the etiology of autoimmune disease.

Aging↗