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Biomedical subjects

D H Lynch

Publications and source records attributed to D H Lynch.

At least 37 records · Page 2Linked to original sources
PubMed1998

CD4 regulates susceptibility to Fas ligand- and tumor necrosis factor-mediated apoptosis.

The current knowledge of CD4 function is limited to its role as a necessary coreceptor in TCR-initiated signaling. We have investigated whether CD4 regulates additional T cell functions. Using human primary resting CD4+ T cells, we demonstrate that CD4 activation is sufficient to induce lymphocyte death. Immediately after CD4 cross-linking, CD4+ T cells are rendered susceptible to apoptosis mediated by TNF or FasL. This, together with the concomitant induction of FasL within the same population, results in significant CD4+ T cell death in vitro. The CD4-dependent induction of susceptibility to apoptosis that is mediated by TNF or FasL is protein synthesis independent but phosphorylation dependent. After CD4 activation, PKC regulates susceptibility to apoptosis mediated by FasL but not the induction of susceptibility to TNF-dependent apoptosis. Moreover, significant differences between CD3 and CD4 activation were observed with regards to the kinetics of induction of CD4+ T cell susceptibility to FasL- and TNF-mediated apoptosis. Altogether, these results provide a model with which to study the molecular mechanisms regulating lymphocyte survival after CD4 activation, and highlight the potential role of CD4 in controlling lymphocyte apoptosis under physiological conditions or in disease states such as HIV infection.

Apoptosis↗
PubMed1998

Human purified protein derivative-specific CD4+ T cells use both CD95-dependent and CD95-independent cytolytic mechanisms.

CTL, both CD4+ and CD8+, are essential in the eradication of intracellular pathogens. Data generated using murine T cells have suggested a critical role for CD95 (Fas, Apo-1) in CD4+ T cell-induced apoptosis of target cells. In contrast, CD8+ CTL predominantly use the perforin/granzyme lytic pathway. At present little is known about the mechanism of CD4+ CTL lytic function during intracellular infection in humans. We have used human CD4+ T cells specific for purified protein derivative (PPD) of Mycobacterium tuberculosis to explore whether CD95 is the dominant cytolytic mechanism. PPD-reactive CD4+ clones efficiently lysed Ag-pulsed autologous monocytes, adherent macrophages, and EBV-transformed B cells. Addition of an antagonistic CD95 Ab had a minimal effect on cytolysis, whereas addition of MgEGTA to block perforin/granzyme resulted in complete inhibition of killing. In contrast, lysis of activated peripheral blood B cells could be partially blocked with the antagonistic CD95 Ab. Supporting these observations, monocytes, macrophages, and EBV-transformed B cells were not lysed by an agonistic CD95 Ab. Activated B cells were readily lysed by the agonistic CD95 Ab. T cell clones triggered through the TCR with anti-CD3 were capable of lysing the CD95-sensitive Jurkat T cell line in a CD95-dependent manner, but were also able to release granzymes. We conclude that human CD4+ T cells are capable of lysing PPD-pulsed targets using both perforin/granzyme and CD95 pathways. The contribution of CD95 is strictly dependent on target cell susceptibility to CD95-mediated killing.

Antibodies, Blocking↗
PubMed1998

TRAIL: a molecule with multiple receptors and control mechanisms.

Apoptosis research is benefiting from bioinformatic approaches to identify new components of the cell death machinery and novel cell death inducers/receptors. Over the past year, knowledge of the system involving TNF-related apoptosis-inducing ligand (TRAIL) and its receptors has increased via genomic database analysis to include four distinct receptors that interact with a single ligand. Currently, these molecules are of major interest due to their potential roles and application in cancer therapy.

Animals↗
PubMed1998

Induction of dendritic cells (DC) by Flt3 Ligand (FL) promotes the generation of tumor-specific immune responses in vivo.

Daily treatment of mice with human Flt3 ligand (FL) induces the production of large numbers of MHC-class II+, CD11c+, DEC205+ dendritic cells (DC) in both lymphoid and nonlymphoid tissues. Because DC play a pivotal role in the induction of immune responses, we evaluated the effects of FL in the augmentation of anti-tumor immune responses in vivo. Treatment with daily subcutaneous injections of FL not only caused complete regression of tumors in a significant proportion of mice challenged with a syngeneic methylcholanthrene-induced fibrosarcoma, but also caused a significant decrease in the tumor growth rate in the remaining mice. The effects of FL were both dose and schedule dependent, induced regression of tumors established as much as 7 days prior to beginning cytokine treatment, and was mediated by both T-cell mediated and non-T cell-mediated mechanisms. Histopathologic evaluation of tumors during and after systemic treatment with FL revealed a dense infiltration of mononuclear and myeloid precursor cells into both the tumor and tissue surrounding the tumor that generally correlated with the degree of regression and resolved after rejection of the tumor. Collectively, the data suggest that FL may be an important cytokine in the generation of effective anti-tumor immune responses in vivo and the treatment of cancer in situ.

Animals↗
PubMed1997

ZAP-70 tyrosine kinase is required for the up-regulation of Fas ligand in activation-induced T cell apoptosis.

Activation-induced cell death (AICD) is initiated by the TCR-dependent up-regulation of Fas ligand (FasL) mRNA. The subsequently generated soluble or cell-associated FasL gene products bind Fas, leading to apoptosis of the T cells. Although TCR stimulation is essential to initiate AICD, little is known about which TCR-initiated second messengers are required for FasL expression. We provide evidence in this work that T cells lacking the tyrosine kinase ZAP-70 are unable to up-regulate FasL and undergo AICD. Transfection of wild-type ZAP-70 into the ZAP-70-deficient T cells restores their sensitivity to TCR-induced apoptosis, whereas transfection of catalytically inactive ZAP-70 does not. These results provide clear evidence that ZAP-70 tyrosine kinase is essential in up-regulating FasL for TCR-induced apoptosis.

Apoptosis↗
PubMed1997

Fas ligand (CD95L) and B7 expression on dendritic cells provide counter-regulatory signals for T cell survival and proliferation.

Activation of T cells is induced efficiently by dendritic cells (DC), but little is known about the role of DC in the regulation of T cell death. In this study, highly purified DC (DEC-205+, MHC class II(high), B7-1+ [CD80+], B7-2high [CD86high], CD40+, CD11c+) grown from normal mouse bone marrow in granulocyte-macrophage CSF + IL-4 were found to express FasL (CD95L) mRNA by reverse transcriptase PCR and to uniformly express FasL by both flow cytometric and immunocytochemical analyses. These cells, but not DC propagated from FasL-deficient (B6.gld) mice, induced dose-dependent increases in DNA fragmentation in Fas+ Jurkat T cells over 18 h coculture. Addition of mouse Fas-Fc fusion protein at the start of the cultures diminished this effect. Even at high relative concentrations, however, B7-2high DC induced only low levels of DNA fragmentation in Con A or alloactivated splenic T cells, as determined by radio- or spectrofluorometric assays and by in situ nick-end labeling. However, in the presence of CTLA4Ig, a molecule that blocks the B7-CD28 costimulatory pathway, DC that failed to stimulate in primary MLR induced markedly augmented levels of apoptosis in alloactivated T cells. CTLA4Ig treatment also increased the level of DNA fragmentation induced by FasL-deficient DC, indicating the existence of additional potential (Fas-independent) pathways of DC-induced T cell death. These findings suggest that the costimulatory (B7-CD28) and T cell death-inducing pathways may play important counter-regulatory roles in dictating the outcome of (allogeneic) DC-T cell interactions.

Animals↗
PubMed1997

Macrophage-dependent apoptosis of CD4+ T lymphocytes from HIV-infected individuals is mediated by FasL and tumor necrosis factor.

Apoptosis of bystander uninfected CD4+ T lymphocytes by neighboring HIV-infected cells is observed in cell culture and in lymphoid tissue of HIV-infected individuals. This study addresses whether antigen-presenting cells such as human macrophages mediate apoptosis of CD4+ T cells from HIV-infected individuals. Uninfected human macrophages, and to a larger degree, HIV-infected macrophages mediate apoptosis of T cells from HIV-infected, but not from uninfected control individuals. This macrophage-dependent killing targets CD4+, but not CD8+ T lymphocytes from HIV-infected individuals, and direct contact between macrophages and lymphocytes is required. Additional analyses indicated that the apoptosis-inducing ligands, FasL and tumor necrosis factor (TNF), mediate this macrophage-induced apoptosis of CD4+ T cells. These results support a role for macrophage-associated FasL and TNF in the selective depletion of CD4+ T cells in HIV-infected individuals.

Antibodies, Monoclonal↗
PubMed1997

Impaired nonrestricted cytolytic activity in systemic lupus erythematosus: involvement of a pathway independent of Fas, tumor necrosis factor, and extracellular ATP that is associated with little detectable perforin.

OBJECTIVE: To determine the cytolytic effector pathway involved in impaired generation of nonrestricted cytolytic activity in systemic lupus erythematosus (SLE). METHODS: Peripheral blood mononuclear cells (PBMC) from normal subjects and SLE patients were stimulated in vitro with anti-CD3 monoclonal antibody (MAb) and interleukin-2 to promote the generation of nonrestricted cytolytic activity. On day 13 of culture, the PBMC were assayed for cytolytic activity against Fas-Daudi cells and Fas+ Jurkat cells. The effects on cytotoxicity of calcium chelation, antagonist anti-Fas MAb, tumor necrosis factor (TNF) alpha and beta, and ATP were measured. Intracellular perforin expression was determined by intracellular staining, and perforin messenger RNA levels were determined by quantitative competitive reverse transcription-polymerase chain reaction. RESULTS: We demonstrated the existence of a cytolytic pathway that is independent of Fas, TNF alpha, TNF beta, and ATP, but is dependent upon extracellular calcium. Despite its calcium dependence, this pathway is associated with low-to-undetectable levels of perforin. CONCLUSION: Impaired generation of nonrestricted cytolytic activity in SLE is likely due to decreased activity of this Fas-, TNF alpha-, TNF beta-, ATP-independent pathway associated with very low levels of perforin.

Adenosine Triphosphate↗
PubMed1997

CD40 ligand inhibits Fas/CD95-mediated apoptosis of human blood-derived dendritic cells.

Dendritic cells (DC) are considered to be the most potent antigen-presenting cells (APC) in the immune system. In this study, we analyzed the regulation of apoptosis of human peripheral blood-derived DC. DC were generated from adherent peripheral blood mononuclear cells that had been cultured for 7 days with granulocyte-macrophage colony-stimulating factor and interleukin-4. These cells displayed phenotypic properties of DC, including dendritic processes, expression of CD1a and lack of expression of CD14, and were very potent at presenting soluble antigens to T cells. Blood-derived DC were demonstrated to express the Fas/CD95 antigen and an agonist antibody to CD95 strongly induced apoptotic cell death in these cells. Soluble trimeric CD40 ligand potently inhibited both CD95-mediated and spontaneous apoptosis in DC. The data suggest that interactions between members of the tumor necrosis factor family of ligands expressed by T cells with their receptors on DC play an important role in the regulation of apoptosis in DC during antigen presentation and may, therefore, regulate the duration of T cell expansion and cytokine production.

Apoptosis↗
PubMed1997

Regulation of Fas and Fas-ligand expression in NK cells by cytokines and the involvement of Fas-ligand in NK/LAK cell-mediated cytotoxicity.

This study demonstrates cytokine-mediated regulation of Fas and Fas-ligand (Fas-L) expression in human NK cells and the involvement of the Fas-L pathway in NK/LAK cytotoxicity. Freshly isolated, high purified human CD56+CD3- NK cells were found to express Fas and Fas-L. Cytokines further increased the Fas expression in the CD56+CD3- NK cells, with interleukin (IL)-2 being the most potent stimulus followed by IL-12, while IL-7 had no effect. IL-2 and IL-7 equally enhanced the Fas expression in the CD56+CD3+ population, while IL-12 had a less pronounced effect. Incubation of the CD56+CD3- NK cells with IL-2, but not with IL-12 and IL-7, led to an upregulation at the Fas-L expression, whereas neither of the cytokines affected the Fas-L expression in the CD56+CD3+ cells. Antagonistic Fas mAb M3 and Fas-IgG1 fusion protein significantly inhibited NK cytotoxicity towards Fas-expressing Jurkat cells, while non-antagonistic Fas mAb M31 and irrelevant CD14-IgG1 fusion protein had no effect. IL-2-generated LAK cells were much more potent than NK cells in exerting the cytotoxic effect on Jurkat cells, which was also partially inhibited to M3 and Fas-IgG1. Thus, human NK and LAK cells express Fas and Fas-L, utilize the Fas-L cytotoxic pathway and enhance the expression of these molecules in response to cytokines.

Antibodies, Monoclonal↗
PubMed1997

Fas-mediated cytotoxicity by intestinal intraepithelial lymphocytes during acute graft-versus-host disease in mice.

BACKGROUND & AIMS: Host-derived intestinal intraepithelial lymphocytes (IELs) increase in number during acute graft-versus-host disease (GVHD) in mice. In the present study, we examined Fas-mediated cytotoxicity by host-derived IELs against Fas-expressing target cells to see whether Fas/Fas ligand (Fas-L) interaction is involved in the pathogenesis of enteropathy during acute GVHD. METHODS: Acute GVHD was induced by injection of parental spleen cells into nonirradiated F1 mice. The expression of Fas antigen on the intestinal epithelial cells (IECs) was examined by flow cytometry, and the expression of messenger RNA (mRNA) for Fas-L, interleukin 2, and interferon gamma in host-derived IELs was assessed by reverse-transcription polymerase chain reaction. Fas-mediated cytotoxicity by host-derived IELs was assessed using Fas-transfected cells, IECs, and Fas immunoglobulin Fc fusion protein (Fas Fc). RESULTS: Fas antigen was constitutively expressed on the cell surface of IECs before and after GVHD induction. Although Fas-L mRNA was not detected or detected scarcely in either alphabeta or gammadelta IELs before GVHD induction, both IELs expressed high levels of mRNA for Fas-L and interferon gamma after GVHD induction. Host-derived IELs during acute GVHD showed cytotoxicity against Fas-transfected target cells and IECs, which was partly blocked by addition of Fas Fc. CONCLUSIONS: Fas/Fas-L-mediated cytotoxicity by host-derived IELs may be partly responsible for the enteropathy during acute GVHD.

Animals↗
PubMed1997

Characterization of poly(glycolide-co-D,L-lactide)/poly(D,L-lactide) microspheres for controlled release of GM-CSF.

PURPOSE: This study describes the preparation and characterization of a controlled release formulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) encapsulated in poly(glycolide-co-D,L-lactide) (PLGA) and poly(D,L-lactide) (PLA) microspheres. METHODS: GM-CSF was encapsulated in PLGA/PLA microspheres by a novel silicone oil based phase separation process. Several different blends of PLGA and low molecular weight PLA were used to prepare the microspheres. The microspheres and the encapsulated GM-CSF were extensively characterized both in vitro and in vivo. RESULTS: Steady release of GM-CSF was achieved over a period of about one week without significant "burst" of protein from the microspheres. Analysis of microsphere degradation kinetics by gel permeation chromatography (GPC) indicated that low molecular weight PLA enhanced the degradation of the PLGA and thereby affected release kinetics. GM-CSF released from the microspheres was found to be biologically active and physically intact by bioassay and chromatographic analysis. Analysis of serum from mice receiving huGM-CSF indicated that the GM-CSF was biologically active and that a concentration of greater than 10 ng/mL was maintained for a period lasting at least nine days. MuGM-CSF was not detected following in vivo administration of muGM-CSF microspheres. The tissues of mice receiving muGM-CSF microspheres were characterized by infiltration of neutrophils, and macrophages which were in significant excess of those found in mice administered with placebo controls (i.e. microspheres without GM-CSF). CONCLUSIONS: This study demonstrates the influence of formulation parameters on the encapsulation of GM-CSF in PLGA/PLA microspheres and its controlled release in biologically active form. The intense local tissue reaction in mice to muGM-CSF microspheres demonstrates the importance of the mode of delivery on the pharmacologic activity of GM-CSF.

Animals↗
PubMed1997

Flt3 ligand induces tumor regression and antitumor immune responses in vivo.

Daily treatment of mice with recombinant human Flt3 ligand (huFlt3L) results in a dramatic numerical increase in the number of dendritic cells (DCs) in vivo. Since DCs are pivotal in the induction of immune responses, we tested whether Flt3L treatment of mice challenged with a syngeneic methylcholanthrene (MCA)-induced fibrosarcoma would augment the generation of effective antitumor immune responses in vivo. Flt3L treatment not only induced complete tumor regression in a significant proportion of mice, but also decreased tumor growth rate in the remaining mice. A preliminary characterization of the cellular mechanisms involved suggests that Flt3L may be important in the treatment of cancer in situ through the generation of specific antitumor immune responses.

Animals↗
PubMed1997

Potential for involvement of Fas antigen/Fas ligand interaction in apoptosis of epithelial cells by intraepithelial lymphocytes in murine small intestine.

Intestinal epithelial cells (i-EC), which move to the villus tips from the crypts, rapidly die by apoptosis at the villus tips and are perpetually renewed at the crypts. To determine whether the Fas antigen (Fas)/Fas ligand (FasL) system is involved in the mechanism leading to apoptosis of i-EC, we examined the expression of Fas and FasL on the i-EC and intestinal intraepithelial lymphocytes (i-IEL) in normal mice. A high level of Fas was expressed on both the i-EC and i-IEL, whereas FasL was expressed in the i-IEL, especially in high-density fraction upon separation (high-density i-IEL), but not in the i-EC. The high-density i-IEL exhibited cytotoxicity against not only Fas transfectant but also the i-EC, and the cytotoxicity was inhibited by addition of Fas-Fragment c chimeric fusion protein. Thus, a significant fraction of i-IEL, such as high-density i-IEL, may partly contribute to induction of apoptosis in the effete i-EC via Fas/FasL interaction.

Animals↗
PubMed1996

Impaired survival and proliferation in IL-7 receptor-deficient peripheral T cells.

Mice genetically deficient in IL-7R(alpha) are highly lymphopenic in the peripheral lymphoid organs. The functional competence of T cells that have developed in the absence of an IL-7R signal was investigated. Three important observations were made using several in vitro activation regimens. First, stimulation of T cells from IL-7R -/- mice at limiting dilution with immobilized Abs to CD3, CD4 or CD8, and CD18 revealed a six- to sevenfold reduction in the frequency of clonogenic T cells compared with T cells from IL-7R +/+ mice. IL-7R -/- T cells were also significantly less responsive to alloantigen as well as to receptor-independent stimuli such as PMA and ionomycin. Furthermore, the average clone size of single IL-7R -/- T cells was 50% smaller than that of IL-7R +/+ T cells. These data suggest that the reduced clonogenicity was predominantly due to intrinsic deficiencies in the ability of IL-7R -/- T cells to proliferate upon stimulation. Second, analysis of the kinetics of cell growth of IL-7R -/- T cells revealed that a significant proportion of T cells failed to proliferate within the first 72 h of in vitro stimulation, with the majority undergoing programmed cell death. Third, both clonogenic IL-7 -/- T cells and IL-7R +/+ T cells showed a similar proliferative response in the presence of IL-2 and similar survival kinetics, indicating that a subpopulation of IL-7R -/- T cells is functionally mature. We propose that an absence of IL-7R signaling not only affects T cell development in the thymus, but also results in the accumulation of functionally inactive T cells in the periphery.

Animals↗
PubMed1996

Intracellular signaling for inducible antigen receptor-mediated Fas resistance in B cells.

CD40 ligand-activated B cells are sensitive targets for CD4+ Th1 effector cells that kill in a Fas-dependent fashion. Susceptibility to apoptosis is counteracted by Ag receptor binding that produces a state of resistance to Fas engagement in otherwise sensitive targets. In the present study, protection from Th1-mediated apoptosis was found to be induced by protein kinase C and calcium signals, which in combination mimicked the level of Fas resistance produced by surface Ig engagement. Signaling for Fas resistance did not alter Fas expression. Furthermore, B cells that were protected against Th1-mediated apoptosis were also resistant to apoptosis mediated by soluble, rFas ligand. Taken together, these results indicate that signaling for protection against Fas-mediated apoptosis does not depend on alteration of the interaction between B cell target and Th1 effector populations. Instead, surface IgM-derived protein kinase C and calcium signals appear to produce an intracellular change in the Fas signaling pathway that develops over a period of hours and interferes with the apoptotic process through a mechanism that depends on protein synthesis.

Animals↗