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K M Murphy

Publications and source records attributed to K M Murphy.

At least 55 records · Page 3Linked to original sources
PubMed1999

Differentiation of the mononuclear phagocyte system during mouse embryogenesis: the role of transcription factor PU.1.

During mouse embryogenesis, macrophage-like cells arise first in the yolk sac and are produced subsequently in the liver. The onset of liver hematopoiesis is associated with the transition from primitive to definitive erythrocyte production. This report addresses the hypothesis that a similar transition in phenotype occurs in myelopoiesis. We have used whole mount in situ hybridization to detect macrophage-specific genes expressed during mouse development. The mouse c-fms mRNA, encoding the receptor for macrophage colony-stimulating factor (CSF-1), was expressed on phagocytic cells in the yolk sac and throughout the embryo before the onset of liver hematopoiesis. Similar cells were detected using the mannose receptor, the complement receptor (CR3), or the Microphthalmia transcription factor (MITF) as mRNA markers. By contrast, other markers including the F4/80 antigen, the macrophage scavenger receptor, the S-100 proteins, S100A8 and S100A9, and the secretory product lysozyme appeared later in development and appeared restricted to only a subset of c-fms-positive cells. Two-color immunolabeling on disaggregated cells confirmed that CR3 and c-fms proteins are expressed on the same cells. Among the genes appearing later in development was the macrophage-restricted transcription factor, PU.1, which has been shown to be required for normal adult myelopoiesis. Mice with null mutations in PU.1 had normal numbers of c-fms-positive phagocytes at 11.5dpc. PU.1(-/-) embryonic stem cells were able to give rise to macrophage-like cells after cultivation in vitro. The results support previous evidence that yolk sac-derived fetal phagocytes are functionally distinct from those arising in the liver and develop via a different pathway.

Animals↗
PubMed1999

Lineage-specific requirement for signal transducer and activator of transcription (Stat)4 in interferon gamma production from CD4(+) versus CD8(+) T cells.

CD4(+) and CD8(+) T cells exhibit important differences in their major effector functions. CD8(+) T cells provide protection against pathogens through cytolytic activity, whereas CD4(+) T cells exert important regulatory activity through production of cytokines. However, both lineages can produce interferon (IFN)-gamma, which can contribute to protective immunity. Here we show that CD4(+) and CD8(+) T cells differ in their regulation of IFN-gamma production. Both lineages require signal transducer and activator of transcription (Stat)4 activation for IFN-gamma induced by interleukin (IL)-12/IL-18 signaling, but only CD4(+) T cells require Stat4 for IFN-gamma induction via the TCR pathway. In response to antigen, CD8(+) T cells can produce IFN-gamma independently of IL-12, whereas CD4(+) T cells require IL-12 and Stat4 activation. Thus, there is a lineage-specific requirement for Stat4 activation in antigen-induced IFN-gamma production based on differences in TCR signaling between CD4(+) and CD8(+) T cells.

Animals↗
PubMed1999

The Ets transcription factor ERM is Th1-specific and induced by IL-12 through a Stat4-dependent pathway.

Interleukin 12 (IL-12)-induced T helper 1 (Th1) development requires Stat4 activation. However, antigen-activated Th1 cells can produce interferon gamma (IFN-gamma) independently of IL-12 and Stat4 activation. Thus, in differentiated Th1 cells, factors regulated by IL-12 and Stat4 may be involved in IFN-gamma production. Using subtractive cloning, we identified ERM, an Ets transcription factor, to be a Th1-specific, IL-12-induced gene. IL-12-induction of ERM occurred in wild-type and Stat1-deficient, but not Stat4-deficient, T cells, suggesting ERM is Stat4-inducible. Retroviral expression of ERM did not restore IFN-gamma production in Stat4-deficient T cells, but augmented IFN-gamma expression in Stat4-heterozygous T cells. Ets factors frequently regulate transcription via cooperative interactions with other transcription factors, and ERM has been reported to cooperate with c-Jun. However, in the absence of other transcription factors, ERM augmented expression of an IFN-gamma reporter by only 2-fold. Thus, determining the requirement for ERM in Th1 development likely will require gene targeting.

Animals↗
PubMed1999

TCR-independent pathways mediate the effects of antigen dose and altered peptide ligands on Th cell polarization.

We examined the role of the peptide/MHC ligand in CD4+ T cell differentiation into Th1 or Th2 cells using a TCR alphabeta transgenic mouse specific for hemoglobin (Hb)(64-76)/I-Ek. We identified two altered peptide ligands of Hb(64-76) that retain strong agonist activity but, at a given dose, induce cytokine patterns distinct from the Hb(64-76) peptide. The ability of these peptides to produce distinct cytokine patterns at identical doses is not due to an intrinsic qualitative property. Each peptide can induce Th2 cytokines at low concentrations and Th1 cytokines at high concentrations and has a unique range of concentrations at which these distinct effects occur. The pattern of cytokines produced from limiting dilution of naive T cells demonstrated that the potential to develop an individual Th1 or Th2 cell is stochastic, independent of Ag dose. We propose that the basis for the observed effects on the Th1/Th2 balance shown by the altered peptide ligands and the amount of Ag dose involves the modification of soluble factors in bulk cultures that are the driving force that polarize the population to either a Th1 or Th2 phenotype.

Animals↗
PubMed1999

Modulation of airway inflammation by passive transfer of allergen-specific Th1 and Th2 cells in a mouse model of asthma.

Although evidence is strong that Th cells play a major role in mediating the airway inflammation observed in asthma, the relative contributions of the Th cell subsets, Th1 and Th2, are unclear. It has been suggested that asthma is driven by Th2 predominant responses in the lung, but other data suggest a role for Th1 cells as well. Here we show by intracellular cytokine staining and flow cytometric analysis that in the murine model of OVA-induced airway inflammation, both Th1 and Th2 cells are recruited to the airways. Th1 cells predominate early in the response and Th2 cells predominate late. We further show that increasing the number of Th1 cells by passive transfer of OVA-specific Th1 cells results in increased inflammation. This effect is observed regardless of whether the T cells are transferred before sensitization or after airway inflammation is already in progress. Transfer of Th1 cells also results in increased recruitment of host T cells of both Th1 and Th2 phenotypes. Passive transfer of Th2 cells results in little change in the inflammatory response. These results demonstrate that Ag-specific Th1 cells are not protective in this model of asthma, but rather may potentiate the inflammatory response.

Adoptive Transfer↗
PubMed1999

Tpm1, a locus controlling IL-12 responsiveness, acts by a cell-autonomous mechanism.

Th phenotype development is controlled not only by cytokines but also by other parameters including genetic background. One site of genetic variation between murine strains that has direct impact on Th development is the expression of the IL-12 receptor. T cells from B10.D2 and BALB/c mice show distinct control of IL-12 receptor expression. When activated by Ag, B10.D2 T cells express functional IL-12 receptors and maintain IL-12 responsiveness. In contrast, under the same conditions, BALB/c T cells fail to express IL-12 receptors and become unresponsive to IL-12, precluding any Th1-inducing effects if subsequently exposed to IL-12. Previously, we identified a locus, which we termed T cell phenotype modifier 1 (Tpm1), on murine chromosome 11 that controls this differential maintenance of IL-12 responsiveness. In this study, we have produced a higher resolution map around Tpm1. We produced and analyzed a series of recombinants from a first-generation backcross that significantly narrows the genetic boundaries of Tpm1. This allowed us to exclude from consideration certain previous candidates for Tpm1, including IFN-regulatory factor-1. Also, cellular analysis of F1(B10.D2 x BALB/c) T cells demonstrates that Tpm1 exerts its effect on IL-12 receptor expression in a cell-autonomous manner, rather than through influencing the extracellular milieu. This result strongly implies that despite the proximity of our locus to the IL-13/IL-4 gene cluster, these cytokines are not candidates for Tpm1.

Alleles↗
PubMed1999

Endogenous galectins and effect of galectin hapten inhibitors on the differentiation of the chick mesonephros.

Galectins are galactoside-binding lectins. In the mesonephros of the chick embryo, the 16-kDa galectin is abundant in the glomerular and tubular basement membranes where it colocalizes with fibronectin and laminin. To test whether galectin-glycoprotein interactions could play a role in mesonephric development, the effects of the galectin hapten inhibitors thiodigalactoside (TDG) and lactose on the differentiation of the cultured mesonephros were investigated. When compared to control saccharide-free or maltose-treated cultures, mesonephroi cultured in the presence of TDG and lactose exhibited defects in tissue organization. These included a distorted tubule shape, pseudo-stratification of the tubular epithelium, and detachment of glomerular podocytes from the basement membrane. The presence of molecular differentiation markers in the developing mesonephros was investigated. In vivo, expression of the epithelial-specific cell adhesion molecule E-cadherin is restricted to differentiated tubular epithelial cells, whereas the intermediate filament protein vimentin is present in mesonephrogenic mesenchyme and is undetectable in tubular epithelial cells. In mesonephroi cultured in the absence of sugars or in the presence of maltose, the expression pattern of these two marker molecules resembles that found in the mesonephros in vivo. In contrast, in the mesonephroi cultured in the presence of TDG and lactose, the epithelial tubular cells expressing E-cadherin also express vimentin. Re-expression of vimentin in the tubular epithelial cells could indicate a partial reversal to a mesenchymal phenotype. Results suggest that galectin-glycoprotein interactions in the basement membrane are important in the maintenance of the renal epithelial phenotype. Dev Dyn 1999;215:248-263.

Animals↗
PubMed1999

Induction of interferon-gamma production in Th1 CD4+ T cells: evidence for two distinct pathways for promoter activation.

IFN-gamma produced by CD4+ T helper 1 (Th1) cells promotes protection against intracellular pathogens. Antigen activation of Th1 cells is an important mode of IFN-gamma induction, but here we analyze a second, antigen-nonspecific pathway capable of inducing full IFN-gamma transcription. IL-12 or IL-18 alone do not induce IFN-gamma mRNA, and only modestly augment antigen-induced IFN-gamma mRNA from Th1 cells. However, IL-12 and IL-18 together fully induce IFN-gamma transcription independently of TCR-activated signals, by a mechanism that does not simply involve Stat4 and NF-kappaB activation, but requires additional protein synthesis. Cyclosporin A inhibits TCR-induced IFN-gamma production, but not IL-12/IL-18-induced IFN-gamma production, biochemically discriminating between these pathways. These results suggest that the two pathways induce IFN-gamma production through functionally segregated but spatially overlapping cis-acting elements, similar to other genes under the control of two or more promoters.

Animals↗
PubMed1999

T helper differentiation proceeds through Stat1-dependent, Stat4-dependent and Stat4-independent phases.

Much of our focus in understanding Th1/Th2 development has been on the signals delivered by IL-12 and IL-4 as final determinants of terminal T cell differentiation. Because extinction of IL-12 signaling in early Th2 development could potentially be important in imprinting a more permanent Th2 phenotype on a population of T cells, we have also examined various parameters regulating the IL-12 signaling pathway. Whereas IL-4 appears to repress functional IL-12 signaling through inhibition of IL-12R beta 2 expression, IFN-gamma in the mouse, and IFN-alpha in the human appear to induce IL-12R beta 2 expression and promote IL-12 responsiveness. We propose that Th1 development can be considered in two stages, capacitance and development. Capacitance would simply involve expression of IL-12R beta 1 and beta 2 subunits, regulated by TCR, IL-4 and IFNs. The second stage, development, we propose is the true IL-12 induced developmental stage, involving expression of Stat4 inducible proteins. In the human, this may also occur via IFN-alpha, which is able to activate Stat4. It is perhaps possible that all of Stat4 actions on Th1 development may be exert directly by Stat4 at the IFN-gamma gene, however we suggest that, more likely, Stat4 may act to induce Th1 development through the induction of other non-cytokine genes, whose stable expression maintains the transcriptional state of a Th1 cell.

Animals↗
PubMed1999

Bcl-2 inhibits early apoptotic events and reveals post-mitotic multinucleation without affecting cell cycle arrest in human epithelial tumor cells exposed to etoposide.

UNLABELLED: Defective apoptotic mechanisms are considered to play a role in both the development of malignancy and resistance to chemotherapeutic drugs. The Bcl-2 family of proteins regulate the cellular commitment to survive or die when challenged with various apoptotic stimuli. PURPOSE: The purpose of this study was to identify the point at which Bcl-2 interrupts the apoptotic cascade initiated following exposure of human tumor cells to etoposide. METHODS: A stable Bcl-2-expressing HeLa-transfected clonal cell line, along with its control-vector-transfected counterpart, were utilized in this study. Following etoposide exposure, cells were examined for cell cycle arrest, formation of hyperdiploid cells, apoptotic DNA degradation, loss of plasma membrane integrity, levels of expression of members of the Bcl-2 protein family, caspase activation, degradation of poly(ADP-ribose) polymerase and movement of Bax from cytosol to cellular membrane fractions. RESULTS: Caspase activation, poly(ADP-ribose) polymerase degradation and Bax membrane insertion were initiated rapidly following etoposide removal, concomitantly with cell cycle arrest. Whereas Bcl-2 had no effect on etoposide-induced cell arrest, it interrupted all aspects of apoptosis, including activation of caspases, poly(ADP-ribose) polymerase degradation, DNA fragmentation and loss of plasma membrane integrity. Surprisingly, Bcl-2 also blocked Bax membrane insertion. In addition, Bcl-2 also prevented the increase in cellular levels of Bak, Bax and Bcl-xL, along with degradation of actin and Bax. However, inhibition of etoposide-induced apoptosis by Bcl-2 resulted in the accumulation of giant, multinucleated cells that eventually lost the ability to exclude trypan blue without apoptotic morphology or DNA degradation. CONCLUSIONS: These results indicate that biochemical apoptotic processes are initiated concomitant with etoposide-induced cell cycle arrest and are interrupted by Bcl-2 overexpression. However, the aberrant mitotic events induced by etoposide are sufficient to kill these cells even in the absence of apoptosis.

Antineoplastic Agents, Phytogenic↗
PubMed1999

Displaced odontoid fracture in a 9-month-old child.

A 9-month-old child sustained a displaced odontoid fracture in a motor vehicle accident. She was treated with closed reduction and a Minerva cast for 6 weeks, followed by a collar for an additional 2 weeks. At 5-year follow-up, she had made a complete recovery with solid union, no deformity, and no instability.

Accidents, Traffic↗
PubMed1999

Immunology at 2000.

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Allergy and Immunology↗
PubMed1999

A description of cutaneous-subcutaneous pythiosis in fifteen dogs.

Information regarding signalment, duration of clinical signs, history of swimming, results of CBC and serum biochemical analyses, biopsy findings and mycological results, together with treatments and outcome, was retrieved from the medical records of 15 dogs with a diagnosis of pythiosis made between 1985 and 1995 at the Colleges of Veterinary Medicine, North Carolina State University and the University of Florida. Most of the dogs were young (median age 22 months) and represented larger breeds (> 20 kg). Lesions were characteristically chronic, ulcerated, and nodular with multiple draining tracts on the limbs, thoracic wall or perineal regions. The median duration of these lesions was 3 months with a range of 2 weeks-6 months. Seven dogs had a history of swimming. Peripheral eosinophilia was observed in 14 of the dogs. Cytological evaluation of discharge, aspirates, or impression smears made from biopsy specimens revealed hyphae in five of 11 dogs (45%). Histopathological evaluation using the Gomori Methenamine-Silver (GMS) stain was the most useful test for providing presumptive evidence of cutaneous pythiosis. Immunotherapy or antifungal therapy using either amphotericin B, liposomal nystatin, itraconazole, or ketoconazole were all unsuccessful. The only dog to survive underwent amputation of the affected limb; thus, the prognosis for cutaneous pythiosis in the dog is poor.

Animals↗
PubMed1999

Expression of the p56(Lck) Y505F mutation in CD45-deficient mice rescues thymocyte development.

Mice deficient in the transmembrane protein tyrosine phosphatase CD45 exhibit a block in thymocyte development. To determine whether the block in thymocyte development was due to the inability to dephosphorylate the inhibitory phosphorylation site (Y505) in p56(lck) (Lck), we generated CD45-deficient mice that express transgenes for the Lck Y505F mutation and the DO11.10 T-cell antigen receptor (TCR). CD4 single-positive T cells developed and accumulated in the periphery. Treatment with antigen resulted in thymocyte apoptosis and the loss of transgenic-TCR-bearing cells. Peripheral CD45-deficient T cells from the mice expressing both transgenes responded to antigen by increasing CD69 expression, interleukin-2 production, and proliferation. These results indicate that thymocyte development requires the dephosphorylation of the inhibitory site in Lck by CD45.

Animals↗
PubMed1998

GATA-3-dependent enhancer activity in IL-4 gene regulation.

Previously, we analyzed the proximal IL-4 promoter in directing Th2-specific activity. An 800-base pair proximal promoter conferred some Th2-selective expression in transgenic mice. However, this region directed extremely low reporter mRNA levels relative to endogenous IL-4 mRNA, suggesting that full gene activity requires additional enhancer elements. Here, we analyzed large genomic IL-4 regions for enhancer activity and interaction with transcription factors. The proximal IL-4 promoter is only moderately augmented by GATA-3, but certain genomic regions significantly enhanced GATA-3 promoter transactivation. Some enhancing regions contained consensus, GATA sites that bound Th2-specific complexes. However, retroviral transduction of GATA-3 into developing T cells induced IL-5 to full Th2 levels, but only partially restored IL-4 production. Thus, we propose that GATA-3 is permissive, but not sufficient, for full IL-4 enhancement and may act through GATA elements surrounding the IL-13/IL-4 gene locus.

Animals↗