Biomedical subjects

T J Ley

Publications and source records attributed to T J Ley.

At least 37 records · Page 2Linked to original sources

PubMed1998

Reduced beta-globin gene expression in adult mice containing deletions of locus control region 5' HS-2 or 5' HS-3.

To gain insights into the functions of individual DNA'se hypersensitive sites within the beta globin locus control region (LCR), we deleted the endogenous 5' HS-2 and HS-3 regions from the mouse germline using homologous recombination techniques. We demonstrated that the deletion of either murine 5' HS-2 or 5' HS-3 reduced the expression of the embryonic epsilon y and beta h1 globin genes minimally in yolk sac-derived erythrocytes, but that both knockouts reduced the output of the adult beta (beta-Major + beta-Minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK-Neo cassette was retained within either the HS-2 or HS-3 region, a much more severe reduction in globin gene expression was observed at all developmental stages. PGK-Neo was shown to be expressed in an erythroid-specific fashion when it was retained in the HS-3 position. These results show that neither 5' HS-2 nor HS-3 is required for the activity of embryonic globin genes, nor are these sites required for correct developmental switching. However, each site is required for approximately 30% of the total LCR activity associated with adult beta-globin gene expression in adult red blood cells. Each site therefore contains some non-redundant information that contributes to adult globin gene function.

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PubMed1998

Stochastic, stage-specific mechanisms account for the variegation of a human globin transgene.

The random insertion of transgenes into the genomic DNA of mice usually leads to widely variable levels of expression in individual founder lines. To study the mechanisms that cause variegation, we designed a transgene that we expected to variegate, which consisted of a beta-globin locus control region 5' HS-2 linked in tandem to a tagged human beta-globin gene (into which a Lac-Z cassette had been inserted). All tested founder lines exhibited red blood cell-specific expression, but levels of expression varied >1000-fold from the lowest to the highest expressing line. Most of the variation in levels of expression appeared to reflect differences in the percentage of cells in the peripheral blood that expressed the transgene, which ranged from 0.3% in the lowest expressing line to 88% in the highest; the level of transgene expression per cell varied no more than 10-fold from the lowest to the highest expressing line. These differences in expression levels could not be explained by the location of transgene integration, by an effect of beta-galactosidase on red blood cell survival, by the half life of the beta-galactosidase enzyme or by the age of the animals. The progeny of all early erythroid progenitors (BFU-E colony-forming cells) exhibited the same propensity to variegate in methylcellulose-based cultures, suggesting that the decision to variegate occurs after the BFU-E stage of erythroid differentiation. Collectively, these data suggest that variegation in levels of transgene expression are due to local, integration site-dependent phenomena that alter the probability that a transgene will be expressed in an appropriate cell; however, these local effects have a minimal impact on the transgene's activity in the cells that initiate transcription.

Age Factors↗

PubMed1998

Production of fully active recombinant murine granzyme B in yeast.

Granzyme B (GzmB) is a neutral serine protease found in cytotoxic lymphocytes; this enzyme is critically involved in delivering the rapid apoptotic signal to susceptible target cells. GzmB has been difficult to study and has not yet been produced in non-mammalian systems because of the complex processing events that are thought to be required for its activation. In this report, we have successfully produced fully active, soluble recombinant GzmB (rGzmB) in a yeast-based system by fusing GzmB cDNA in frame with yeast alpha-factor cDNA, using the yeast KEX2 signal peptidase to release the processed enzyme into the supernatant of yeast cultures. We expressed the proenzyme form of GzmB as well and determined that pro-GzmB is efficiently converted to its active form by the cysteine proteinase dipeptidyl peptidase I. The fully processed enzyme was able to hydrolyze the synthetic substrate N-t-butyloxycarbonyl-L-alanyl-L-alanyl-L-aspartyl (Boc-Ala-Ala-Asp) thiobenzyl ester with a kcat of 17 s-1 and catalytic efficiency kcat/Km of 181,237 M-1 S-1; the recombinant enzyme is therefore at least twice as active as purified native GzmB. In addition, the recombinant enzyme hydrolyzes Boc-Ala-Ala-Met thiobenzyl ester with a kcat of 3.2 S-1 and a catalytic efficiency kcat/Km of 65,306 M-1 S-1. Purified rGzmB can also cleave the putative substrate caspase-3 into its signature p20/p10 forms. Unlike caspases, rGzmB is not sensitive to inhibition by several peptide-based inhibitors, including Ac-DEVD-CHO, Ac-YVAD-CMK, and ZIETD-FMK, as well as Zn2+ (a known inhibitor of caspase-3). Structural studies of rGzmB may allow us to better understand the substrate specificity of this enzyme and to design better inhibitors.

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PubMed1998

Augmented inflammatory responses and altered wound healing in cathepsin G-deficient mice.

BACKGROUND: Cathepsin G is a neutral serine proteinase that exists primarily in azurophilic granules of neutrophils, but also as a proteolytically active membrane-bound form. While the specificity and many in vitro biological activities have been described for cathepsin G, little is known about the role of this enzyme in neutrophil function in vivo, particularly as it applies to the wound-healing process. OBJECTIVE: To determine the role of cathepsin G in cutaneous tissue repair by examination of full-thickness incisional wound healing in mice with a null mutation for cathepsin G. METHODS: Paired, full-thickness linear incisions were made on the backs of cathepsin G +/+ and cathepsin G -/- mice, and wound tissue was harvested at days 1, 2, 3, 5, 7, 10, and 14 after wounding. Neutrophil influx, myeloperoxidase activity, and migration were examined using light microscopy, the myeloperoxidase assay, and modified Boyden chamber technique, respectively. Wound-breaking strength was measured using tensiometry. RESULTS: The absence of cathepsin G led to a 42% decrease in wound-breaking strength at day 7 after wounding (n=28; P<.002), which returned to the level of control mice by day 10 after wounding. Wound tissue sections in mice lacking cathepsin G also showed a 26% increase in neutrophil myeloperoxidase activity (n=12; P=.001) and an 18% increase in neutrophil influx (n=14; P=.002) at day 3 after wounding. Wound fluid collected on day 5 after wounding from cathepsin G-deficient mice attracted 58% more neutrophils than wound fluid collected from control mice (n=4; P<.05). CONCLUSIONS: Neutrophil cathepsin G is important during the early inflammatory stage of wound healing. Cathepsin G may be involved in processing 1 (or more) soluble mediator(s) in the wound milieu that is responsible for neutrophil chemotaxis. Our findings suggest that tight regulation of inflammation is necessary to prevent impaired healing during early tissue repair.

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PubMed1998

How do cytotoxic lymphocytes kill their targets?

CD8+ cytotoxic lymphocytes, natural killer cells and lymphokine-activated killer cells depend primarily on the perforin/granzyme system to kill their targets, while CD4+ T cells utilize Fas and other mechanisms to induce cell death. The molecular mechanisms used by these pathways to induce target cell apoptosis may converge on common death substrates.

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PubMed1998

Mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis.

Neutrophil elastase (NE) is a potent serine proteinase whose expression is limited to a narrow window during myeloid development. In neutrophils, NE is stored in azurophil granules along with other serine proteinases (cathepsin G, proteinase 3 and azurocidin) at concentrations exceeding 5 mM. As a result of its capacity to efficiently degrade extracellular matrix, NE has been implicated in a variety of destructive diseases. Indeed, while much interest has focused on the pathologic effects of this enzyme, little is known regarding its normal physiologic function(s). Because previous in vitro data have shown that NE exhibits antibacterial activity, we investigated the role of NE in host defense against bacteria. Generating strains of mice deficient in NE (NE-/-) by targeted mutagenesis, we show that NE-/- mice are more susceptible than their normal littermates to sepsis and death following intraperitoneal infection with Gram negative (Klebsiella pneumoniae and Escherichia coli) but not Gram positive (Staphylococcus aureus) bacteria. Our data indicate that neutrophils migrate normally to sites of infection in the absence of NE, but that NE is required for maximal intracellular killing of Gram negative bacteria by neutrophils.

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PubMed1997

Perforin/granzyme-dependent and independent mechanisms are both important for the development of graft-versus-host disease after murine bone marrow transplantation.

Graft-versus-host disease (GvHD) is the major limiting toxicity of allogeneic bone marrow transplantation. T cells are important mediators of GvHD, but the molecular mechanisms that they use to induce GvHD are controversial. Three effector pathways have been described for cytotoxic T lymphocytes: one requires perforin and granzymes, the second Fas (APO-1; CD95) and its ligand. Thirdly, secreted molecules (e.g., TNF-alpha, gamma-IFN) can also mediate cytotoxicity. Together, these mechanisms appear to account for virtually all cytotoxicity induced by activated CTL in standard in vitro lytic assays. Using transplants across histocompatibility barriers, we were able to analyze the contributions of these effector molecules to cell-mediated cytotoxicity in vivo in a GvHD model. We found that Fas ligand is an important independent mediator of class II-restricted acute murine GvHD, while perforin/granzyme-dependent mechanisms have only a minor role in that compartment. In contrast, perforin/ granzyme-dependent mechanisms are required for class I-restricted acute murine GvHD, while Fas ligand is not. The perforin/granzyme pathway may therefore represent a novel target for anti-GvHD drug design. In support of this approach, we provide additional data suggesting that specific perforin/granzyme inhibitors should not adversely affect hematopoietic recovery after transplantation.

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PubMed1997

Residual cytotoxicity and granzyme K expression in granzyme A-deficient cytotoxic lymphocytes.

Cytotoxic lymphocytes contain granules that have the ability to induce apoptosis in susceptible target cells. The granule contents include perforin, a pore-forming molecule, and several granzymes, including A and B, which are the most abundant serine proteases in these granules. Granzyme B-deficient cytotoxic T lymphocytes (CTL) have a severe defect in their ability to rapidly induce apoptosis in their targets, but have an intact late cytotoxicity pathway that is in part perforin-dependent. In this report, we have created mice that are deficient for granzyme A and characterized their phenotype. These mice have normal growth and development and normal lymphocyte development, activation, and proliferation. Granzyme A-deficient CTL have a small but reproducible defect in their ability to induce 51Cr and 125I-UdR release from susceptible allogeneic target cells. Since other granzyme A-like tryptases could potentially account for the residual cytotoxicity in granzyme A-deficient CTL, we cloned the murine granzyme K gene, which is linked to granzyme A in humans, and proved that it is also tightly linked with murine granzyme A. The murine granzyme K gene (which encodes a tryptase similar to granzyme A) is expressed at much lower levels than granzyme A in CTL and LAK cells, but its expression is unaltered in granzyme A-/- mice. The minimal cytotoxic defect in granzyme A-/- CTL could be due to the existence of an intact, functional early killing pathway (granzyme B dependent), or to the persistent expression of additional granzyme tryptases like granzyme K.

Amino Acid Sequence↗

PubMed1997

Mechanisms responsible for granzyme B-independent cytotoxicity.

Using granzyme B-deficient mice obtained by gene targeting, we previously demonstrated that granzyme B is required for the rapid induction of apoptotic target cell death by cytotoxic T lymphocytes (CTLs); however, CTLs are also equipped with additional effector mechanisms. In the present study, we examined the mechanisms responsible for granzyme B-independent cytotoxicity using in vitro lytic assays with CTLs derived from mice deficient for both granzyme B and Fas ligand (FasL) (granzyme B-/- x gld/gld) or for perforin and FasL (perforin x gld/gld). Our results show that primary mixed lymphocyte reaction (MLR)-derived CTLs from granzyme B-/- x gld/gld mice induce apoptosis of allogeneic targets with less efficiency and a longer delay than CTLs deficient for granzyme B alone. The residual cytotoxicity in granzyme B-/- x gld/gld CTLs is primarily accounted for by a perforin-dependent mechanism, since perforin-/- x gld/gld CTLs have virtually no residual cytotoxic activity in our assays. Granzyme B-independent cytotoxicity is therefore partially accounted for by the Fas pathway and partially by another perforin-dependent mechanism.

Animals↗

PubMed1997

Molecular cloning, chromosomal localization, and expression of murine dipeptidyl peptidase I.

Dipeptidyl peptidase I (DPPI) is a lysosomal cysteine protease that catalyzes the sequential removal of dipeptides from the amino termini of various protein substrates. We have isolated a cDNA coding for murine DPPI from mouse thymus and spleen cDNA libraries. The deduced amino acid sequence codes for a protein of 462 amino acid residues; comparison of this deduced sequence with that of rat and human DPPI revealed 90.1% and 77.8% identity, respectively. Using DPPI cDNA, we obtained two BAC (Bacterial Artificial Chromosome) clones that contained the murine DPPI locus. The DPPI gene consists of seven exons and 6 introns, and spans approximately 20 kilobases. Using fluorescence in situ chromosome hybridization, we localized murine DPPI to chromosome 7D3-E1.1. We determined that DPPI protein is widely distributed in mouse tissues, although its relative abundance varies from tissue to tissue. In contrast to previous reports, we show here that DPPI mRNA and protein levels and enzymatic activity are unchanged during in vitro T cell activation, implying that this enzyme is not rate-limiting for granzyme processing.

Amino Acid Sequence↗

PubMed1997

Altered myeloid development and acute leukemia in transgenic mice expressing PML-RAR alpha under control of cathepsin G regulatory sequences.

Acute promyelocytic leukemia (APML) is characterized by abnormal myeloid development, resulting an accumulation of leukemic promyelocytes that are often highly sensitive to retinoic acid. A balanced t(15;17) (q22;q21) reciprocal chromosomal translocation is found in approximately 90% of APML patients; this translocation fuses the PML gene on chromosome 15 to the retinoic acid receptor alpha (RAR alpha) gene on chromosome 17, creating two novel fusion genes, PML-RAR alpha and RAR alpha-PML. The PML-RAR alpha fusion gene product, which is expressed in virtually all patients with t(15;17), is thought to play a direct role in the pathogenesis of APML. To determine whether PML-RAR alpha is sufficient to cause APML in an animal model, we used the promyelocyte-specific targeting sequences of the human cathepsin G (hCG) gene to direct the expression of a PML-RAR alpha cDNA to the early myeloid cells of transgenic mice. Mice expressing the hCG-PML-RAR alpha transgene were found to have altered myeloid development that was characterized by increased percentages of immature and mature myeloid cells in the peripheral blood, bone marrow, and spleen. In addition, approximately 30% of transgene-expressing mice eventually developed acute myeloid leukemia after a long latent period. The splenic promyelocytes of mice with both the nonleukemic and leukemic phenotypes responded to all-trans retinoic acid (ATRA) treatment, which caused apoptosis of myeloid precursors. Although low-level expression of the hCG-PML-RAR alpha transgene is not sufficient to directly cause acute myeloid leukemia in mice, its expression alters myeloid development, resulting in an accumulation of myeloid precursors that may be susceptible to cooperative transforming events.

Animals↗

PubMed1997

The role of granzyme B cluster proteases in cell-mediated cytotoxicity.

Granzymes are neutral serine proteases that are stored in the specialized lytic granules of cytotoxic lymphocytes. A mutation introduced into the granzyme B locus leads to a severe defect in the ability of cytotoxic lymphocytes to induce apoptosis in susceptible target cells, and reduces the severity of class I-dependent acute graft-versus-host disease (GvHD). However, granzyme B-independent cytotoxicity also exists: in CD8+ cells, most of it is perforin-dependent, but in CD4+ cells, the Fas system and an additional pathway are involved. The identification of these pathways and their physiological relevance may lead to new approaches for inhibiting cytotoxic lymphocyte functions.

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PubMed1996

Long-range disruption of gene expression by a selectable marker cassette.

Recent studies have suggested that the retention of selectable marker cassettes (like PGK-Neo, in which a hybrid gene consisting of the phosphoglycerate kinase I promoter drives the neomycin phosphotransferase gene) in targeted loci can cause unexpected phenotypes in "knockout" mice due to disruption of expression of neighboring genes within a locus. We have studied targeted mutations in two multigene clusters, the granzyme B locus and the beta-like globin gene cluster. The insertion of PGK-Neo into the granzyme B gene, the most 5' gene in the granzyme B gene cluster, severely reduced the normal expression of multiple genes within the locus, even at distances greater than 100 kb from the mutation. Similarly, the insertion of a PGK-Neo cassette into the beta-globin locus control region (LCR) abrogates the expression of multiple globin genes downstream from the cassette. In contrast, a targeted mutation of the promyelocyte-specific cathepsin G gene (which lies just 3' to the granzyme genes in the same cluster) had minimal effects on upstream granzyme gene expression. Although the mechanism of these-long distance effects are unknown, the expression of PGK-Neo can be "captured" by the regulatory domain into which it is inserted. These results suggest that the PGK-Neo cassette can interact productively with locus control regions and thereby disrupt normal interactions between local and long-distance regulatory regions within a tissue-specific domain.

Animals↗

PubMed1996

Cleavage of CPP32 by granzyme B represents a critical role for granzyme B in the induction of target cell DNA fragmentation.

Cytotoxic T lymphocytes (CTLs) are able to recognize and destroy target cells bearing foreign antigen using one of two distinct mechanisms: granule- or Fas-mediated cytotoxicity. The exact mechanisms involved in the induction of apoptotic cell death remain elusive; however, it seems likely that a family of cysteine proteases related to interleukin-1beta converting enzyme are involved. One family member, CPP32, has been identified as an intracellular substrate for granzyme B, a CTL-specific serine protease responsible for the early induction of target cell DNA fragmentation. Here we use cytolytic cells from granzyme B-deficient mice to confirm that cleavage and activation of CPP32 represents a nonredundant role for granzyme B and that this activation plays a role in the induction of DNA fragmentation in target cells, a signature event for apoptotic cell death. A peptide inhibitor of CPP32-like proteases confirmed the function of these enzymes in fragmentation. 51Cr release was not suppressed under these conditions, suggesting that granzyme B cleavage of CPP32 is primarily involved in the induction of DNA fragmentation and not membrane damage during CTL-induced apoptosis.

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PubMed1996

Metalloelastase is required for macrophage-mediated proteolysis and matrix invasion in mice.

Macrophages secrete a variety of proteinases that are thought to participate in remodeling of the extracellular matrix associated with inflammatory processes. We have eliminated expression of the macrophage metalloelastase (MME) gene by targeted disruption to assess the role of this protein in macrophage-mediated proteolysis. We found that the macrophages of MME-deficient (MME-/-) mice have a markedly diminished capacity to degrade extracellular matrix components. In addition, MME-/- macrophages are essentially unable to penetrate reconstituted basement membranes in vitro and in vivo. MME is therefore required for macrophage-mediated extracellular matrix proteolysis and tissue invasion.

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PubMed1996

The role of granzyme B in murine models of acute graft-versus-host disease and graft rejection.

A complete molecular description of the syndromes of graft-versus-host disease (GVHD) and graft rejection could have a significant impact on clinical bone marrow transplantation. Recent in vitro experiments (Heusel et al, Cell 76:977, 1994 and Shresta et al, Proc Natl Acad Sci USA 92:5679, 1995) have shown that the putative mediators of these two syndromes, cytotoxic lymphocytes (CTL) and natural killer (NK) cells, respectively, initiate a program of cell death (apoptosis) in susceptible target tissues in a manner critically dependent on the serine protease Granzyme B (gzm B). In the present study, we have analyzed the phenotype of gzm B-deficient mice using experimental transplant models designed to isolate their CD8+ CTL, CD4+ CTL, and NK compartments. We found a significant impairment in class I-dependent GVHD mediated by gzm B -/- CD8+ CTL, whereas class II-dependent GVHD was not altered using gzm B -/- CD4+ effectors. In a hybrid resistance model, gzm B -/- hosts rejected haplo-identical marrow grafts as efficiently as did their wild-type littermates. This result is surprising in light of a severe defect in the ability of gzm B -/- NK cells to induce apoptosis in susceptible targets in vitro. These in vivo data define significant role for gzm B in cytotoxicity mediated by CD8+ CTL, but not by CD4+ CTL. Furthermore, these results do not support a model of hybrid resistance in which NK cells play a pivotal role.

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PubMed1996

Regulation of azurophil granule-associated serine protease genes during myelopoiesis.

During myelopoiesis, the primary or azurophil granules are synthesized at the promyelocyte stage of development. The biogenesis of these granules requires the coordinated regulation of a large number of genes that comprise the constituent proteins of the granules themselves. The genes encoding the granule-associated proteins appear to be regulated primarily at the level of gene transcription; that is, most of the granule genes appear to be transcriptionally activated at the beginning of the promyelocyte stage and are transcriptionally repressed at the transition to myelocytes. The cis-acting DNA elements and trans-regulatory factors that control the regulation of granule genes are just beginning to be defined. The granule-associated genes are dispersed throughout the genome; however, several of the serine protease genes expressed in these granules are tightly clustered and the tight clustering may play an important role for their regulation. Recent transgenic studies suggest that DNA elements required for myeloid-specific targeting may be located just upstream from or very near to the granule genes themselves. DNA elements similar to the globin locus control region will probably play a role in the expression of these genes, although locus control elements have not yet been defined for any azurophil granule genes. Future studies directed at defining these key regulatory elements will enhance our understanding of the molecular events that underlie myeloid development.

Azure Stains↗

PubMed1996

Analysis of mice containing a targeted deletion of beta-globin locus control region 5' hypersensitive site 3.

To examine the function of murine beta-globin locus region (LCR) 5' hypersensitive site 3 (HS3) in its native chromosomal context, we deleted this site from the mouse germ line by using homologous recombination techniques. Previous experiments with human 5' HS3 in transgenic models suggested that this site independently contains at least 50% of total LCR activity and that it interacts preferentially with the human gamma-globin genes in embryonic erythroid cells. However, in this study, we demonstrate that deletion of murine 5' HS3 reduces expression of the linked embryonic epsilon y- and beta H 1-globin genes only minimally in yolk sac-derived erythroid cells and reduces output of the linked adult beta (beta major plus beta minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK-neo cassette was left within the HS3 region of the LCR, a much more severe phenotype was observed at all developmental stages, suggesting that PGK-neo interferes with LCR activity when it is retained within the LCR. Collectively, these results suggest that murine 5' HS3 is not required for globin gene switching; importantly, however, it is required for approximately 30% of the total LCR activity associated with adult beta-globin gene expression in adult erythrocytes.

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