Biomedical subjects

T D Randall

Publications and source records attributed to T D Randall.

14 recordsLinked to original sources

PubMed1998

Mice deficient for the ecto-nicotinamide adenine dinucleotide glycohydrolase CD38 exhibit altered humoral immune responses.

CD38 is a membrane-associated ecto-nicotinamide adenine dinucleotide (NAD+) glycohydrolase that is expressed on multiple hematopoietic cells. The extracellular domain of CD38 can mediate the catalysis of NAD+ to cyclic adenosine diphosphoribose (cADPR), a Ca2+-mobilizing second messenger, adenosine diphosphoribose (ADPR), and nicotinamide. In addition to its enzymatic properties, murine CD38 has been shown to act as a B-cell coreceptor capable of modulating signals through the B-cell antigen receptor. To investigate the in vivo physiological function(s) of this novel class of ectoenzyme we generated mice carrying a null mutation in the CD38 gene. CD38-/- mice showed a complete loss of tissue-associated NAD+ glycohydrolase activity, showing that the classical NAD+ glycohydrolases and CD38 are likely identical. Although murine CD38 is expressed on hematopoietic stem cells as well as on committed progenitors, we show that CD38 is not required for hematopoiesis or lymphopoiesis. However, CD38-/- mice did exhibit marked deficiencies in antibody responses to T-cell-dependent protein antigens and augmented antibody responses to at least one T-cell-independent type 2 polysaccharide antigen. These data suggest that CD38 may play an important role in vivo in regulating humoral immune responses.

ADP-ribosyl Cyclase↗

PubMed1998

Characterization of a population of cells in the bone marrow that phenotypically mimics hematopoietic stem cells: resting stem cells or mystery population?

We have identified a population of cells in murine bone marrow that has many of the phenotypic characteristics attributed to resting hematopoietic stem cells but does not reconstitute irradiated mice. These cells express high levels of Sca-1, H-2K and CD38 and low levels of Thy-1.1, but do not express CD34 nor any of the lineage markers including CD3, CD4, CD5, CD8 NK1.1, I-A, B220, Ig(MGA), CD40, kappa, Mac-1, Gr-1 or Ter119. In addition, this population can be found at normal frequency in nu/nu as well as rag-1-/- mice. These cells incorporate only low levels of Rh123, are resistant to the cytotoxic effects of 5-fluorouracil and, consistent with their resting phenotype, less than 2% of these cells are in the S/G2/M phases of the cell cycle. The only phenotypic characteristic that distinguishes these cells from the lineage- Sca-1+, Thy-1.1low long-term reconstituting hematopoietic stem cell population is their lack of c-kit expression. Here we have explored the possibility that these cells represent a truly resting population of hematopoietic stem cells. We found that the lineage-, Sca-1+, c-kit- cells do not respond to hematopoietic growth factors in vitro, either alone or in combination with stromal layers. Furthermore, these cells do not form in vivo spleen colonies nor do they have the ability to reconstitute irradiated mice. Thus, this population may represent either a population of resting stem cells for which we lack the appropriate activating stimulus, or simply represent a "mystery population" that phenotypically mimics most of the physical properties of resting stem cells. Given the close phenotypic similarity of the c-kit- mystery population cells to the c-kit+ long-term reconstituting stem cells, investigators must be rigorous to exclude their effects from other stem cell assays.

ADP-ribosyl Cyclase↗

PubMed1998

Arrest of B lymphocyte terminal differentiation by CD40 signaling: mechanism for lack of antibody-secreting cells in germinal centers.

Despite extensive research, the role of CD40 signaling in B cell terminal differentiation remains controversial. Here we show that CD40 engagement arrests B cell differentiation prior to plasma cell formation. This arrest is manifested at a molecular level as a reduction in mRNA levels of secretory immunoglobulin gene products such as mu(s) and J chain as well as the loss of the transcriptional regulator BLIMP-1. Furthermore, the inhibition of B cell differentiation by CD40 engagement could not be overcome by either mitogens or cytokines, but could be reversed by antibodies that interfere with the CD40/gp39 interaction. These data suggest that secretory immunoglobulin is not produced by B cells that are actively engaged by gp39-expressing T cells.

Animals↗

PubMed1998

CD38: a new paradigm in lymphocyte activation and signal transduction.

CD38 is a type II transmembrane glycoprotein that is extensively expressed on cells of hematopoietic and non-hematopoietic lineage. Although the intracellular domain of CD38 is not homologous to any known proteins, the extracellular domain of CD38 is structurally related to enzymes in the ADP-ribosyl cyclase family. The structural homology between CD38 and the cyclase family members extends to functional homology, as the extracellular domain of CD38 can mediate the catalysis of beta-NAD+ into nicotinamide, ADP-ribose (ADPR) and, to a lesser extent, into cyclic ADPR-ribose (cADPR). Extensive investigation in other systems has shown that cADPR is an important regulator of intracellular Ca2+ release. Since engagement of CD38 on hematopoietic cells with anti-CD38 Abs has been shown to have potent effects on a number of in vitro cellular responses, we have speculated that cADPR might control CD38-mediated signal transduction. However, it has been difficult to understand how a mediator which is typically an intracellular signaling molecule could potentiate its effects from an extracellular location, thus posing a dilemma which pertains to all ecto-enzymes and the mechanisms by which they regulate signal transduction and cellular processes. This review describes the biologic properties of murine CD38, its role in humoral immunity, and its signal transduction properties in B lymphocytes. We suggest that signaling through CD38 represents a new paradigm in lymphocyte signal transduction and is predicated upon extracellular, rather than intracellular, crosstalk.

ADP-ribosyl Cyclase↗

PubMed1997

Phenotypic and functional changes induced at the clonal level in hematopoietic stem cells after 5-fluorouracil treatment.

A significant fraction of hematopoietic stem cells (HSCs) have been shown to be resistant to the effects of cytotoxic agents such as 5-fluorouracil (5-FU), which is thought to eliminate many of the rapidly dividing, more committed progenitors in the bone marrow and to provide a relatively enriched population of the most primitive hematopoietic progenitor cells. Although differences between 5-FU-enriched progenitor populations and those from normal bone marrow have been described, it remained unclear if these differences reflected characteristics of the most primitive stem cells that were revealed by 5-FU, or if there were changes in the stem-cell population itself. Here, we have examined some of the properties of the stem cells in the bone marrow before and after 5-FU treatment and have defined several activation-related changes in the stem-cell population. We found that long-term reconstituting stem cells decrease their expression of the growth factor receptor c-kit by 10-fold and increase their expression of the integrin Mac-1 (CD11b). These changes begin as early as 24 hours after 5-FU treatment and are most pronounced within 2 to 3 days. This activated phenotype of HSCs isolated from 5-FU-treated mice is similar to the phenotype of stem cells found in the fetal liver and to the phenotype of transiently repopulating progenitors in normal bone marrow. We found that cell cycle is induced concomitantly with these physical changes, and within 2 days as many as 29% of the stem-cell population is in the S/G2/M phases of the cell cycle. Furthermore, when examined at a clonal level, we found that 5-FU did not appear to eliminate many of the transient, multipotent progenitors from the bone marrow that were found to be copurified with long-term repopulating, activated stem cells. These results demonstrate the sensitivity of the hematopoietic system to changes in its homeostasis and correlate the expression of several important surface molecules with the activation state of HSCs.

Animals↗

PubMed1996

Expression of murine CD38 defines a population of long-term reconstituting hematopoietic stem cells.

Using a monoclonal antibody to murine CD38, we showed that a population of adult bone marrow cells that expressed the markers Sca-1 and c-kit but lacked the lineage markers Mac-1, GR-1, B220, IgM, CD3, CD4, CD8 and CD5 could be subdivided by the expression of CD38. We showed that CD38high c-kit+ Sca-1+, linlow/-cells sorted from adult bone marrow cultured with interleukin-3 (IL-3), IL-6, and kit-L produced much larger colonies in liquid culture at a greater frequency than their CD38low/- counterparts. In addition, we found that CD36low/ - cells contained most of the day-12 colony-forming units-spleen (CFU-S) but were not long-term reconstituting cells, whereas the population that expressed higher levels of CD38 contained few, but significant, day-12 CFU-S and virtually all the long-term reconstituting stem cells. Interestingly, the CD38high Sca-1+ c-kit+ linlow/- cells isolated from day-E14.5 fetal liver were also found to be long-term reconstituting stem cells. This is in striking contrast to human hematopoietic progenitors in which the most primitive hematopoietic cells from fetal tissues lack the expression of CD38. Furthermore, because antibodies to CD38 could functionally replace antibodies to Thy-1.1 in a stem cell purification procedure, the use of anti-CD38 may be more generally applicable to the purification of hematopoietic stem cells from mouse strains that do not express the Thy-1.1 allele.

ADP-ribosyl Cyclase↗

PubMed1994

Mechanism and subcellular localization of secretory IgM polymer assembly.

The predominant functions of secreted IgM, complement fixation and transcytosis across epithelial barriers via the poly(Ig) receptor, are properties of IgM polymers; thus, the mechanisms that regulate oligomerization are critical to immune function. We have developed methods to assess the mechanism and subcellular location of IgM polymer formation. Using denaturing agarose/SDS-polyacrylamide gel electrophoresis and sucrose gradient fractionation, we demonstrate that IgM polymers are assembled in a stepwise fashion in which primary intermediates, including heavy chain-light chain complexes, are sequentially incorporated. Assembly intermediates include both covalent and non-covalent components, suggesting that IgM subunit interactions precede covalent assembly. IgM polymers are assembled from primary intermediates containing immature N-linked oligosaccharides. Polymerization is insensitive to brefeldin A and occurs at temperatures that inhibit protein transport along the secretory pathway. Together, these data demonstrate that IgM polymerization occurs early in the secretory pathway, most likely in the endoplasmic reticulum. These results are consistent with a model in which secretory IgM represents an oligomeric protein that is retained in the endoplasmic reticulum and does not mature along the secretory pathway until complete assembly is achieved.

Animals↗

PubMed1994

IgM hexamers?

There has been a universal tendency to regard IgM antibodies as pentameric molecules comprising five immunoglobulin monomeric subunits joined by a single J-chain protein. Is this the only form of secreted IgM, or are the possibilities more complex? In this article, Joseph Brewer and colleagues propose that the IgM polymers secreted in primary immune responses may be more heterogeneous than previously believed and that, as a consequence, the biological activity of IgM may have considerable flexibility.

Animals↗

PubMed1993

MHC class II limits the functional expression of endogenous superantigens in B cells.

The open reading frames of the 3' long terminal repeat of mouse mammary tumor viruses (MMTV) encode superantigens. When expressed with MHC class II molecules on the surface of B cells, superantigens stimulate T cells expressing receptors with particular V beta elements. By using B cell lymphomas as model systems, we demonstrate that stimuli, including certain lymphokines (IL-2 and IL-5) and LPS, that lead to increased MMTV transcript levels in B cells did not increase functional superantigen presentation. Furthermore, stimulation of BCL1 and CH12 cells with LPS resulted in a pronounced reduction in superantigen presentation, even though MMTV transcript levels were increased. In contrast to these effects, functional superantigen expression was increased under conditions in which class II levels were increased, even when MMTV levels remained constant, indicating that the levels of newly synthesized class II are important for functional superantigen expression. These data together imply that the amount of cell surface class II limits superantigen presentation and/or that superantigens can associate only with a limited pool of class II.

Animals↗

PubMed1993

Interleukin-5 (IL-5) and IL-6 define two molecularly distinct pathways of B-cell differentiation.

Interleukin-5 (IL-5) and IL-6 have both been reported to act as B-cell differentiation factors by stimulating activated B cells to secrete antibody. However, it has not been possible to directly compare the effects of these two lymphokines because of the lack of a suitable B-cell line capable of responding to both. We have identified a clonal, inducible B-cell lymphoma, CH12, that has this property. Both IL-5 and IL-6 can independently stimulate increases in steady-state levels of immunoglobulin and J-chain mRNA and proteins, and they both induce the differentiation of CH12 into high-rate antibody-secreting cells. Nevertheless, there are significant differences in the activities of these two lymphokines. First, while IL-6 acts only as a differentiation factor, IL-5 also augments the proliferation of CH12 cells. Second, the differentiation stimulated by IL-5 but not by IL-6 is partially inhibited by IL-4. Inhibition of IL-5-induced differentiation was not at the level of IL-5 receptor expression, since IL-4 did not inhibit IL-5-induced proliferation. Third, IL-5 but not IL-6 stimulated increased mouse mammary tumor proviral gene expression in CH12 cells. These results demonstrate that while both IL-5 and IL-6 may act as differentiation factors for B cells, they induce differentiation by using at least partially distinct molecular pathways. Our results also establish that B cells characteristic of a single stage of development can independently respond to IL-4, IL-5, and IL-6.

Animals↗

PubMed1992

Direct evidence that J chain regulates the polymeric structure of IgM in antibody-secreting B cells.

IgM is secreted in two functional polymeric forms. Secreted IgM was originally thought to be exclusively a pentameric molecule containing J (joining) chain, but many B cells also secrete hexameric IgM lacking J chain. Hexameric IgM may play an important role in the immune system, since it is up to 20 times more active than pentameric IgM in initiating the complement cascade. The predominant polymeric form of IgM secreted by B cell lines, either pentameric or hexameric, correlates with the concentration of J chain present during polymerization, and cells that express high levels of J chain secrete mostly IgM pentamers. The B cell lymphoma WEHI-231 does not express J chain, and the majority of its secreted IgM is polymerized as hexamers. When a J chain-encoding cDNA was expressed in these cells, the secreted IgM was found to be almost exclusively pentameric. However, although the expression of J chain dramatically altered the phenotype of the IgM secreted by these cells, it had little effect on their secretory rate. We conclude that J chain regulates the structure and function of the IgM polymers secreted by B cells, but it is not necessary for either IgM polymerization or secretion.

Amino Acid Sequence↗

PubMed1992

J chain synthesis and secretion of hexameric IgM is differentially regulated by lipopolysaccharide and interleukin 5.

Two functional polymeric forms of IgM can be produced by antibody-secreting B cells. Hexameric IgM lacks detectable J (joining) chain and activates complement 17-fold better than pentameric IgM, which usually contains one J chain per pentamer. Using the inducible B-cell lymphoma CH12, we determined if the synthesis of a particular polymeric form of IgM is a fixed property of B cells or can be altered. Lipopolysaccharide (LPS)-stimulated CH12 cells produced mixtures of IgM hexamers and pentamers, resulting in antibody with high complement-fixing activity. In contrast, interleukin-5-stimulated CH12 cells secreted predominantly pentameric IgM, with a correspondingly lower lytic activity. Differences in lytic activity were due only to the amount of hexameric IgM in the secreted antibody. Interleukin 5 stimulated higher production of J chain RNA and protein than LPS, while LPS induced the highest levels of the secretory form of mu protein. The amount of hexameric IgM secreted was therefore inversely proportional to the level of intracellular J chain protein in the responding B cells. We conclude that the biologic function of IgM produced by B cells differs depending on how they are stimulated and that this difference may be regulated by the relative availabilities of J chain and secretory mu proteins during IgM polymerization.

Animals↗

PubMed1992

Mouse mammary tumor proviral gene expression in cells of the B lineage.

Mouse mammary tumor proviruses (MMTV) use a common enhancer/promoter region to accommodate their transcription in two different cellular environments. In mammary tissue, transcription is regulated through the hormone response element located in the 5' LTR. In B cells, transcription is hormone independent, can be stimulated following B cell activation, and is distinct from the transcription of other known inducible genes, including immunoglobulin. The open reading frame (ORF) in the viral 3' LTR has at least two functions. Its gene product(s) acts as a viral superantigen, but also has autoregulatory properties, leading to MMTV transactivation. We propose a scheme suggesting that MMTV evolved to use the B cell as an intermediary in its viral life cycle.

Animals↗

PubMed1990

The biological effects of IgM hexamer formation.

The inducible B cell lymphoma, CH12, and its in vitro adapted subclone, CH12-LBK, produce immunoglobulins of identical sequence, specificity and isotype, with equivalent affinities for the hapten trimethyl ammonium. However, the hemolytic efficiencies of the antibody secreted by the two cell lines are quite different. Antibody preparations from lipopolysaccharide-stimulated CH12 cells lyse erythrocytes six- to ten times more effectively than antibody preparations of the same concentration from CH12-LBK cells. Both cell lines secrete polymeric IgM, but while CH12-LBK cells secrete predominantly the canonical pentameric IgM, CH12 cells secrete a mixture of pentamers and hexamers. High-efficiency complement-dependent cytolysis is associated with hexameric IgM, which has a specific activity that is approximately twenty times higher than that of the pentameric form. J chain protein is found in the secreted IgM of both cell lines, but is associated only with the pentameric IgM and not with the hexameric form, nor with any intermediate polymers smaller than a pentamer. A deficit in, or the inaccessibility of, J chain protein appears to facilitate hexamer formation. These experiments confirm previously published data showing that J chain is not necessary either for assembly or secretion of polymeric IgM, and suggest instead that J chain may be important in regulating the lytic efficiency of polymeric IgM by controlling the IgM pentamer/hexamer ratio. The experiments further suggest a mechanism, in addition to isotype switching and somatic mutation, by which the biological efficiency of antibodies from a single clone of B cells can be regulated.

Hemolysis↗