ABSTRACT
Biological control of insects by predators may be indirectly influenced by management practices that change the invertebrate community in agroecosystems. In this study we examined effects that mulching and weeding have on predatory beetles (Carabidae and Staphylinidae) and their potential prey in a highbush blueberry field. We compared beetle communities in unweeded control plots to those that were weeded and/or received a single application of compost or pine needle mulch. Compost mulch and weeding significantly affected the carabid community while the staphylinid community responded to compost and pine needle mulches. Effects because of mulch tended to intensify in the year after mulch application for both families. Estimates of species richness and diversity for Carabidae and Staphylinidae were similar in all plot types, but rarefaction curves suggested higher Carabidae richness in unmulched plots despite fewer individuals captured. Carnivorous Carabidae, dominated by Pterostichus melanarius, were most frequently captured in compost plots both years, and omnivores were most frequently captured in unweeded compost. Density of millipedes, the most abundant potential prey, was generally greater in mulched plots, whereas seasonal abundance of small earthworms varied among mulch types. Our results have potential implications for biological control in mulched highbush blueberries depending on beetle consumption rates for key pests and how rates are affected by alternative prey.
Subject(s)
Biodiversity , Blueberry Plants , Coleoptera , Soil , Animals , Feeding Behavior , Food Chain , Population DensityABSTRACT
Control of blueberry maggot, Rhagoletis mendax Curran, typically is achieved with insecticides targeting adult flies before females oviposit in ripening fruit. Management strategies targeting other life stages have received less attention. We tested effects of compost or pine needle mulches on emergence of blueberry maggot flies under laboratory and field conditions. Few flies emerged from pupae that were buried under 20 cm of pine needles in all experiments, but burial in 20 cm of compost did not always result in low fly emergence. Burial of pupae in 5 cm of compost or pine needles did not reduce fly emergence compared with 1 cm in soil. Low emergence with increased mulch depth appeared to be primarily because of failure of flies to ascend to the surface after they exited puparia. Low emergence also was associated with high moisture levels causing rotten, discolored pupae, particularly in the laboratory in compost. No flies emerged from pupae buried in 1 cm of pine needles in the field. In this case no flies exited puparia, likely because high temperatures (>30°C) at the surface killed pupae. Thus, mulch application under highbush blueberries (Vaccinium corymbosum L.) after maggots drop from berries can reduce emergence success of flies from buried pupae, but the level of control will depend on mulch depth and may vary with rainfall and temperature.
Subject(s)
Blueberry Plants , Insect Control , Soil , Tephritidae/physiology , Animals , Larva/physiology , Life Cycle Stages , Pupa/physiology , Temperature , Time FactorsABSTRACT
The feasibility of assessing copper accumulation in agricultural soils using avoidance tests with a Canadian strain of Folsomia candida was investigated under laboratory conditions. The avoidance response to nominal copper sulfate concentrations of 0, 200, 800, 1600 and 3200 mg kg⻹ in OECD soil was inconsistent between trials with the standard plastic cup or a modified Petri dish method requiring less soil. However, combined results from three Petri dish trials decreased variability and provided a 75% avoidance level, close to the 80% criterion proposed for avoidance tests. A Copper avoidance EC50s of 18 mg kg⻹ was obtained using the Petri dish method whether tests were conducted with or without light. While Petri dish tests have potential as a cheap tool to distinguish metal contaminated soils from uncontaminated soils they would be unsuitable for tracking or quantifying changes in metal concentrations. throughout remediation. Advantages and limitations of the method have been presented.
Subject(s)
Copper/pharmacology , Insecta/drug effects , Animals , Behavior, Animal , Biological Assay , Copper/analysis , Soil Pollutants/analysis , Soil Pollutants/pharmacologyABSTRACT
The abundance of the Colorado potato beetle, Leptinotarsa decemlineata (Say), in organically grown potato did not change significantly in response to increasing rates of dehydrated poultry manure. However, peaks of abundance of larvae were shifted forward in time in response to the high rate of organic fertilizer. Tests using excised foliage showed that the shift was not caused by differential larval mortality or longer developmental times. Time allocation to resting, walking, and feeding by adults was similar regardless of fertilizer rate. Adult foliage consumption was unaffected by organic fertilizer rates in no choice tests and significantly affected in few choice tests. A 22% longer larval development time on plants treated with low fertilizer rate than on plants with high rate was the most significant effect. Even though maximum plant height, canopy, biomass, and yield were significantly smaller in the organic than in conventional plots, the suitability of the plants was not affected except for reduced feeding by summer beetles. Summer adults spent less time feeding and consumed two to five times less foliage on organic potato than on inorganically fertilized and conventionally produced plants. The overall influence of fertilizer on Colorado potato beetle populations was limited and therefore can only play a secondary role in management strategies for organic potato. Avoidance of excessive organic fertilizer that promotes short larval development time and extension of the period over which large Colorado potato beetle larvae are present should be recommended.
Subject(s)
Coleoptera/physiology , Fertilizers , Solanum tuberosum/growth & development , Animals , Feeding Behavior , Population Dynamics , Time FactorsABSTRACT
Perennial forages may be ideally suited for fertilization with slow N release amendments such as composts, but difficulties in predicting N supply from composts have limited their routine use in forage production. A field study was conducted to compare the yield and protein content of a binary legume-grass forage mixture and a grass monocrop cut twice annually, when fertilized with diverse composts. In all three years from 1998-2000, timothy (Phleum pratense L.)-red clover (Trifolium pratense L.) and timothy swards were fertilized with ammonium nitrate (AN) at up to 150 and 300 kg N ha(-1) yr(-1), respectively. Organic amendments, applied at up to 600 kg N ha(-1) yr(-1) in the first two years only, included composts derived from crop residue (CSC), dairy manure (DMC), or sewage sludge (SSLC), plus liquid dairy manure (DM). Treatments DM at 150 kg N ha(-1) yr(-1) and CSC at 600 kg N ha(-1) yr(-1) produced cumulative timothy yields matching those obtained for inorganic fertilizer. Apparent nitrogen recovery (ANR) ranged from 0.65% (SSLC) to 15.1% (DMC) for composts, compared with 29.4% (DM) and 36.5% (AN). The legume component (approximately 30%) of the binary mixture acted as an effective "N buffer" maintaining forage yield and protein content consistently higher, and within a narrower range, across all treatments. Integrating compost utilization into livestock systems that use legume-grass mixtures may reduce the risk of large excesses or deficits of N, moderate against potential losses in crop yield and quality, and by accommodating lower application rates of composts, reduce soil P and K accumulation.
Subject(s)
Animal Feed , Models, Theoretical , Nitrogen/analysis , Nitrogen/pharmacokinetics , Soil Pollutants/analysis , Soil Pollutants/pharmacokinetics , Animals , Animals, Domestic , Biological Availability , Fertilizers , Humidity , Phleum/growth & development , Trifolium/growth & developmentABSTRACT
Infection of T cells with HIV-1 induces apoptosis and modulates apoptosis regulatory molecules. Similar effects occur following treatment of cells with individual HIV-1 encoded proteins. While HIV-1 protease is known to be cytotoxic, little is known of its effect on apoptosis and apoptosis regulatory molecules. The ability of HIV-1 protease to kill cells, coupled with the degenerate substrate specificity of HIV-1 protease, suggests that HIV-1 protease may activate cellular factor(s) which, in turn, induce apoptosis. We demonstrate that HIV-1 protease directly cleaves and activates procaspase 8 in T cells which is associated with cleavage of BID, mitochondrial release of cytochrome c, activation of the downstream caspases 9 and 3, cleavage of DFF and PARP and, eventually, to nuclear condensation and DNA fragmentation that are characteristic of apoptosis. The effect of HIV-1 protease is not seen in T cell extracts which have undetectable levels of procaspase 8, indicating a specificity and requirement for procaspase 8.
Subject(s)
Caspases/metabolism , Cytochrome c Group/metabolism , Enzyme Precursors/metabolism , HIV Protease/metabolism , HIV-1/enzymology , Apoptosis/physiology , Caspase 3 , Caspase 8 , Caspase 9 , DNA Fragmentation/physiology , HeLa Cells , Humans , Jurkat Cells , Mitochondria/metabolismABSTRACT
Because the persistence of human immunodeficiency virus (HIV) in cellular reservoirs presents an obstacle to viral eradication, we evaluated whether tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) induces apoptosis in such reservoirs. Lymphocytes and monocyte-derived macrophages (MDM) from uninfected donors do not die following treatment with either leucine zipper human TRAIL (LZhuTRAIL) or agonistic anti-TRAIL receptor antibodies. By contrast, such treatment induces apoptosis of in vitro HIV-infected MDM as well as peripheral blood lymphocytes from HIV-infected patients, including CD4(+) CD45RO(+) HLA-DR(-) lymphocytes. In addition, LZhuTRAIL-treated cells produce less viral RNA and p24 antigen than untreated controls. Whereas untreated cultures produce large amounts of HIV RNA and p24 antigen, of seven treated CD4(+) CD45RO(+) HLA-DR(-) cell cultures, viral RNA production was undetectable in all, p24 antigen was undetectable in six, and proviral DNA was undetectable in four. These data demonstrate that TRAIL induces death of cells from HIV-infected patients, including cell types which harbor latent HIV reservoirs.
Subject(s)
Apoptosis/drug effects , CD4-Positive T-Lymphocytes/drug effects , HIV Infections/immunology , Macrophages/drug effects , Membrane Glycoproteins/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Antiretroviral Therapy, Highly Active , Apoptosis Regulatory Proteins , CD4-Positive T-Lymphocytes/physiology , HIV Infections/drug therapy , HIV Infections/virology , Humans , Immunologic Memory , Jurkat Cells , Macrophages/physiology , RNA, Viral/analysis , Receptors, TNF-Related Apoptosis-Inducing Ligand , Receptors, Tumor Necrosis Factor/analysis , Receptors, Tumor Necrosis Factor/physiology , TNF-Related Apoptosis-Inducing LigandABSTRACT
Apoptosis of macrophages may be a pathogen-directed mechanism of immune escape or may represent appropriate host response to infection. Human monocyte-derived macrophages (MDMs) from healthy donors (C-MDMs) exhibited low-level constitutive apoptosis, but culture of MDMs with opsonized serotype I Streptococcus pneumoniae (I-MDMs) for 20 h resulted in significantly increased apoptosis. I-MDM apoptosis was associated with phagocytosis of bacteria and intracellular killing that was blocked by the caspase inhibitor z-VAD-fmk but not by Fas-blocking antibody. Paraformaldehyde-fixed I-MDMs induced apoptosis in uninfected syngeneic monocytes at levels greater than those in monocytes incubated alone or incubated with fixed C-MDMs. Apoptosis of syngeneic monocytes was blocked by anti-Fas antibody. The immune response of macrophages to S. pneumoniae includes a novel form of apoptosis that is associated with successful phagocytosis and bacterial killing. This response in vivo may regulate the inflammatory response to infection during a successful host response against S. pneumoniae.
Subject(s)
Apoptosis/physiology , Macrophages/immunology , Macrophages/microbiology , Phagocytosis/immunology , Streptococcus pneumoniae/immunology , Amino Acid Chloromethyl Ketones/pharmacology , Apoptosis/drug effects , Caspase Inhibitors , Cells, Cultured , Cysteine Proteinase Inhibitors/pharmacology , Humans , In Situ Nick-End Labeling , Macrophages/drug effects , Monocytes/cytology , Monocytes/immunology , Phagocytosis/drug effects , Reference Values , Regression Analysis , Time FactorsABSTRACT
Infection with the human immunodeficiency virus (HIV) is associated with a progressive decrease in CD4 T-cell number and a consequent impairment in host immune defenses. Analysis of T cells from patients infected with HIV, or of T cells infected in vitro with HIV, demonstrates a significant fraction of both infected and uninfected cells dying by apoptosis. The many mechanisms that contribute to HIV-associated lymphocyte apoptosis include chronic immunologic activation; gp120/160 ligation of the CD4 receptor; enhanced production of cytotoxic ligands or viral proteins by monocytes, macrophages, B cells, and CD8 T cells from HIV-infected patients that kill uninfected CD4 T cells; and direct infection of target cells by HIV, resulting in apoptosis. Although HIV infection results in T-cell apoptosis, under some circumstances HIV infection of resting T cells or macrophages does not result in apoptosis; this may be a critical step in the development of viral reservoirs. Recent therapies for HIV effectively reduce lymphoid and peripheral T-cell apoptosis, reduce viral replication, and enhance cellular immune competence; however, they do not alter viral reservoirs. Further understanding the regulation of apoptosis in HIV disease is required to develop novel immune-based therapies aimed at modifying HIV-induced apoptosis to the benefit of patients infected with HIV.
Subject(s)
Apoptosis , HIV Infections/immunology , HIV/immunology , Lymphocytes/physiology , Lymphocytes/virology , B-Lymphocytes/pathology , B-Lymphocytes/physiology , B-Lymphocytes/virology , HIV Infections/pathology , Humans , Lymphocytes/pathology , Macrophages/pathology , Macrophages/physiology , Macrophages/virology , T-Lymphocytes/pathology , T-Lymphocytes/physiology , T-Lymphocytes/virologyABSTRACT
Dendritic cells (DC) are potent APCs that can be characterized in the murine spleen as CD11b(high)CD11c(high) or CD11b(low)CD11c(high). Daily injection of mice of Flt3 ligand (FL) into mice transiently expands both subsets of DC in vivo, but the effect of administration of GM-CSF on the expansion of DC in vivo is not well defined. To gain further insight into the role of GM-CSF in DC development and function in vivo, we treated mice with polyethylene glycol-modified GM-CSF (pGM-CSF) which has an increased half-life in vivo. Administration of pGM-CSF to mice for 5 days led to a 5- to 10-fold expansion of CD11b(high)CD11c(high) but not CD11b(low)CD11c(high) DC. DC from pGM-CSF-treated mice captured and processed Ag more efficiently than DC from FL-treated mice. Although both FL- and pGM-CSF-generated CD11b(high)CD11c(high) DC were CD8alpha-, a greater proportion of these DC from pGM-CSF-treated mice were 33D1+ than from FL-treated mice. CD11b(low)CD11c(high) DC from FL-treated mice expressed high levels of intracellular MHC class II. DC from both pGM-CSF- and FL-treated mice expressed high levels of surface class II, low levels of the costimulatory molecules CD40, CD80, and CD86 and were equally efficient at stimulating allogeneic and Ag-specific T cell proliferation in vitro. The data demonstrate that treatment with pGM-CSF in vivo preferentially expands CD11b(high)CD11c(high) DC that share phenotypic and functional characteristics with FL-generated CD11b(high)CD11c(high) DC but can be distinguished from FL-generated DC on the basis of Ag capture and surface expression of 33D1.
Subject(s)
Dendritic Cells/cytology , Dendritic Cells/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Integrin alphaXbeta2/biosynthesis , Macrophage-1 Antigen/biosynthesis , Membrane Proteins/physiology , Polyethylene Glycols/pharmacology , Animals , Antigen Presentation , B7-1 Antigen/biosynthesis , Biomarkers , CD40 Antigens/biosynthesis , Cell Division/immunology , Dendritic Cells/metabolism , Dextrans/immunology , Dextrans/metabolism , Female , Granulocyte-Macrophage Colony-Stimulating Factor/administration & dosage , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacokinetics , Half-Life , Hematopoiesis/immunology , Histocompatibility Antigens Class II/biosynthesis , Injections, Intravenous , Intracellular Fluid/immunology , Intracellular Fluid/metabolism , Ligands , Lymphocyte Activation/immunology , Membrane Proteins/administration & dosage , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Knockout , Mice, Transgenic , Ovalbumin/immunology , Ovalbumin/metabolism , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/deficiency , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Recombinant Proteins , T-Lymphocytes/immunologyABSTRACT
The ligand for the receptor tyrosine kinase fms-like tyrosine kinase 3 (flt3), also referred to as fetal liver kinase-2 (flk-2), has an important role in hematopoiesis. The flt3 ligand (flt3L) is a growth factor for hematopoietic progenitors and induces hematopoietic progenitor and stem cell mobilization in vivo. In addition, when mice are treated with flt3L immature B cells, natural killer (NK) cells and dendritic cells (DC) are expanded in vivo. To further elucidate the role of flt3L in hematopoiesis, mice lacking flt3L (flt3L-/-) were generated by targeted gene disruption. Leukocyte cellularity was reduced in the bone marrow, peripheral blood, lymph nodes (LN), and spleen. Thymic cellularity, blood hematocrit, and platelet numbers were not affected. Significantly reduced numbers of myeloid and B-lymphoid progenitors were noted in the BM of flt3L-/- mice. In addition a marked deficiency of NK cells in the spleen was noted. DC numbers were also reduced in the spleen, LN, and thymus. Both myeloid-related (CD11c(++) CD8alpha(-)) and lymphoid-related (CD11c(++) CD8alpha(+)) DC numbers were affected. We conclude that flt3L has an important role in the expansion of early hematopoietic progenitors and in the generation of mature peripheral leukocytes.
Subject(s)
B-Lymphocytes/cytology , Dendritic Cells/cytology , Hematopoiesis/physiology , Hematopoietic Stem Cells/immunology , Killer Cells, Natural/cytology , Membrane Proteins/physiology , Animals , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , Bone Marrow/immunology , Colony-Forming Units Assay , Dendritic Cells/drug effects , Dendritic Cells/immunology , Genomic Library , Hematopoiesis/genetics , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Humans , Interleukin-7/pharmacology , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Kinetics , Leukocytes/cytology , Ligands , Lymph Nodes/immunology , Lymphocyte Culture Test, Mixed , Membrane Proteins/deficiency , Membrane Proteins/genetics , Membrane Proteins/pharmacology , Mice , Mice, Inbred C57BL , Mice, Knockout , Poly I-C/pharmacology , Recombinant Proteins/pharmacology , Spleen/immunology , Thymus Gland/immunologyABSTRACT
The relevance of activation-induced cell death (AICD) of CD4+ T cells to AIDS pathogenesis is unknown. The present study investigates the relationship of AICD to a defined molecular mechanism regulating peripheral T cell homeostasis, Fas-mediated apoptosis, and clinical correlates of the pathogenesis of AIDS. Increased pokeweed mitogen (PWM)-induced AICD (22.8 versus 4.4%, p = 0.006) and Fas-mediated apoptosis (27.7 versus 12.0%, p = 0.002) of CD4+ T cells were observed in HIV+ versus HIV- individuals. Similarly, increased PWM-mediated AICD (16.2 versus 2.2%, p < 0.001) and Fas-mediated apoptosis (25.8 versus 7.6%, p = 0.005) were noted in CD8+ T cells from HIV+ versus HIV- individuals. PWM-induced AICD of CD4+ T cells was blocked (83% median specific inhibition) by Fas-blocking antibodies, whereas PWM-induced AICD of CD8+ T cells was Fas independent. Comparison between PWM- and anti-CD3-mediated AICD of CD4+ T cells indicated that PWM- and not CD3-induced AICD is Fas dependent. HIV+ individuals with an HIV RNA copy number of <30,000 copies/ml had lower PWM-induced AICD of CD4+ T cells than did those with an HIV RNA copy number of >30,000 copies/ml (5.7 versus 22.1%, p = 0.034), and PWM-induced AICD inversely correlated with CD4+ T cell count (R = -0.567, p = 0.043). Initiation of HAART decreased PWM-induced CD4+ T cell AICD from 24.4 to 9.4% posttreatment (p = 0.035). These results demonstrate that PWM-induced AICD of CD4+ T cells from HIV+ individuals is mediated by Fas/FasL, parallels the in vivo susceptibility of the CD4+ T cell to Fas-mediated apoptosis, and correlates with clinical markers of AIDS pathogenesis and response to HAART.
Subject(s)
Apoptosis , CD4-Positive T-Lymphocytes/physiology , HIV Seropositivity/immunology , HIV Seropositivity/virology , HIV , Lymphocyte Activation , fas Receptor/physiology , Anti-HIV Agents/therapeutic use , Biomarkers/analysis , CD4 Lymphocyte Count , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/physiology , Cohort Studies , HIV/immunology , HIV/isolation & purification , HIV Seronegativity , HIV Seropositivity/drug therapy , Humans , Immunity, Cellular , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Pokeweed Mitogens/pharmacology , Polymerase Chain Reaction , RNA, Viral/analysis , Viral LoadABSTRACT
Fas (CD95, APO-1) is a member of the TNF receptor family, and engagement of Fas by its ligand, Fas ligand (FasL), can induce apoptotic death of Fas expressing cells. Signaling through Fas has previously been shown to induce apoptosis of CD34+ human hematopoietic progenitor cells after exposure to IFN-gamma or TFN-alpha. In contrast, we found that FasL promoted a significantly increased viability of primitive CD34+CD38- cells. Thus, incubation with FasL for 48 hours reduced cell death from 46 to 29% compared to cells cultured in medium alone as measured by propidium iodide (PI) incorporation (n = 8, p < 0.02). Inhibition of apoptosis was confirmed by morphological analysis and by the Nicoletti technique. Furthermore, by using a delayed addition assay at the single cell level we found that sFasL treatment had a direct viability-promoting effect on CD34(+)CD38(-) cells. The effect of sFasL was completely blocked by NOK-1, a neutralizing mAb against FasL. In agreement with previous reports, FasL alone slightly increased cell death of more mature CD34(-)CD38+ cells, indicating an interesting shift in the responsiveness to FasL during early hematopoiesis.
Subject(s)
Antigens, CD , Apoptosis/drug effects , Hematopoietic Stem Cells/drug effects , Membrane Glycoproteins/pharmacology , ADP-ribosyl Cyclase , ADP-ribosyl Cyclase 1 , Adult , Antigens, CD34/analysis , Antigens, Differentiation/analysis , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Cell Survival , Fas Ligand Protein , Hematopoietic Cell Growth Factors/pharmacology , Hematopoietic Stem Cells/cytology , Humans , Jurkat Cells , NAD+ Nucleosidase/analysis , Phenotype , Recombinant Fusion Proteins/pharmacology , fas Receptor/physiologyABSTRACT
Daily treatment of mice with fms-like tyrosine kinase 3 ligand (Flt3L) leads to a significant increase in the number of dendritic cells and induces antitumor immunity. Here, we show that Flt3L and CD40 ligand (CD40L) synergize in the generation of immune responses against two poorly immunogenic tumors, leading to complete tumor rejection in a high proportion of mice. Rechallenge of the Flt3L + CD40L-treated mice with the immunizing tumor resulted in complete inhibition of tumor growth, indicating that these animals had developed long-lasting antitumor immunity. In addition, we demonstrate that endogenous CD40L plays a critical role in antitumor immunity, since blockade of CD40-CD40L interactions in vivo prevents the generation of antitumor immunity in therapeutic and vaccination protocols. Dendritic cells generated in mice treated with Flt3L alone or in combination with CD40L were equally potent in stimulating allogeneic T cells and expressed similar levels of MHC class II, CD80, and CD86. However, mice treated with Flt3L + CD40L had significantly more dendritic cells than mice treated with either of the cytokines alone, suggesting that CD40L promotes the proliferation and/or survival of dendritic cells generated by Flt3L treatment. Dendritic cells generated in this manner are likely to be involved in the priming of antitumor immune responses.
Subject(s)
Dendritic Cells/cytology , Dendritic Cells/immunology , Membrane Glycoproteins/administration & dosage , Membrane Glycoproteins/physiology , Membrane Proteins/administration & dosage , Membrane Proteins/physiology , Sarcoma, Experimental/immunology , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/physiology , Animals , CD40 Antigens/physiology , CD40 Ligand , Cell Count , Cell Division/immunology , Cells, Cultured , Coculture Techniques , Dendritic Cells/metabolism , Drug Synergism , Female , Histocompatibility Antigens Class II/biosynthesis , Humans , Injections, Subcutaneous , Interleukin-12/biosynthesis , Interleukin-12/genetics , Ligands , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Knockout , Neoplasm Transplantation , RNA, Messenger/biosynthesis , Sarcoma, Experimental/genetics , Spleen/cytology , Spleen/immunology , Spleen/metabolism , Up-Regulation/immunologyABSTRACT
T cells from HIV infected patients undergo spontaneous apoptosis at a faster rate than those from uninfected patients, are abnormally susceptible to activation induced cell death (AICD), and undergo increased apoptosis in response to Fas receptor ligation. These observations have led to the hypothesis CD4 T cell apoptosis may be a mechanism of CD4 T cell depletion and the pathogenesis of AIDS. Successful treatment of HIV infected patients is accompanied by quantitative and qualitative improvements in immune function reflecting at least partial reversibility of the underlying pathogenesis of HIV. In this report we correlate improvements in markers of immune function with a decrease in apoptosis, and changes in its regulation. Therapy with nelfinavir plus saquinavir in combination with two nucleoside analogue inhibitors of reverse transcriptase dramatically reduces plasma viremia and increases CD4 T cell counts. Coincident with these improvements, CD38 and HLA-DR coexpression on both CD4 and CD8 T cells decrease, and CD45RA and CD62L coexpression increase. Furthermore, spontaneous apoptosis decreases in both CD4 and CD8 T cells (CD4 apoptosis 17.4 vs 2.6%, P=0.005; CD8 apoptosis 15.0 vs 1.0%, P<0.001), as does both Fas mediated apoptosis (CD4 apoptosis 19.0 vs 3.5%, P=0.03; CD8 apoptosis 13.7 vs 1.5%, P=0.002) and CD3 induced AICD (CD4 apoptosis 13.7 vs 3.2%, P=0.001; CD8 apoptosis 29 vs 2.2%, P=0.08). Changes in apoptosis are not associated with changes in Fas receptor expression, but are significantly correlated with changes in activation marker profiles. Although this suggests a possible regulatory role for the apoptosis inhibitory protein FLIP, direct assessment did not reveal quantitative differences in FLIP expression between apoptosis resistant PBL's from HIV negative patients, and apoptosis sensitive PBL's from HIV positive patients. These findings support the hypothesis that apoptosis mediates HIV induced CD4 T cell depletion, but indicate the need for further studies into the molecular regulation of HIV induced apoptosis.
Subject(s)
Apoptosis , HIV Infections/drug therapy , HIV Infections/immunology , Intracellular Signaling Peptides and Proteins , Adolescent , Adult , Anti-HIV Agents/therapeutic use , CASP8 and FADD-Like Apoptosis Regulating Protein , Carrier Proteins/biosynthesis , Drug Therapy, Combination , HIV Infections/virology , HIV Protease Inhibitors/therapeutic use , Humans , Middle Aged , Nelfinavir/therapeutic use , Nucleosides/therapeutic use , Reverse Transcriptase Inhibitors/therapeutic use , Saquinavir/therapeutic use , T-Lymphocytes/immunology , fas Receptor/biosynthesisABSTRACT
Activation-induced cell death of peripheral T cells results from the interaction between Fas and Fas ligand. Resting peripheral T cells are resistant to Fas-induced apoptosis and become susceptible only after their activation. We have investigated the molecular mechanism mediating the sensitization of resting peripheral T cells to Fas-mediated apoptosis following TCR stimulation. TCR activation decreases the steady state protein levels of FLIP (FLICE-like inhibitory protein), an inhibitor of the Fas signaling pathway. Reconstitution of intracellular FLIP levels by the addition of a soluble HIV transactivator protein-FLIP chimera completely restores resistance to Fas-mediated apoptosis in TCR primary T cells. Inhibition of IL-2 production by cyclosporin A, or inhibition of IL-2 signaling by rapamycin or anti-IL-2 neutralizing Abs prevents the decrease in FLIP levels and confers resistance to Fas-mediated apoptosis following T cell activation. Using cell cycle-blocking agents, we demonstrate that activated T cells arrested in G1 phase contain high levels of FLIP protein, whereas activated T cells arrested in S phase have decreased FLIP protein levels. These findings link regulation of FLIP protein levels with cell cycle progression and provide an explanation for the increase in TCR-induced apoptosis observed during the S phase of the cell cycle.
Subject(s)
Apoptosis/immunology , Carrier Proteins/metabolism , Cell Cycle/immunology , Intracellular Signaling Peptides and Proteins , fas Receptor/physiology , Amino Acid Sequence , Apoptosis/drug effects , CASP8 and FADD-Like Apoptosis Regulating Protein , Carrier Proteins/antagonists & inhibitors , Cells, Cultured , Cyclosporine/pharmacology , Down-Regulation/drug effects , Down-Regulation/immunology , Humans , Immune Sera/pharmacology , Immunity, Innate , Interleukin-2/antagonists & inhibitors , Interleukin-2/biosynthesis , Interleukin-2/immunology , Lymphocyte Activation/drug effects , Molecular Sequence Data , Receptors, Antigen, T-Cell/physiology , Signal Transduction/drug effects , Signal Transduction/immunology , Sirolimus/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
mAbs were generated against the extracellular domain of the four known TNF-related apoptosis-inducing ligand (TRAIL) receptors and tested on a panel of human melanoma cell lines. The specificity of the mAb permitted a precise evaluation of the TRAIL receptors that induce apoptosis (TRAIL-R1 and -R2) compared with the TRAIL receptors that potentially regulate TRAIL-mediated apoptosis (TRAIL-R3 and -R4). Immobilized anti-TRAIL-R1 or -R2 mAbs were cytotoxic to TRAIL-sensitive tumor cells, whereas tumor cells resistant to recombinant TRAIL were also resistant to these mAbs and only became sensitive when cultured with actinomycin D. The anti-TRAIL-R1 and -R2 mAb-induced death was characterized by the activation of intracellular caspases, which could be blocked by carbobenzyloxy-Val-Ala-Asp (OMe) fluoromethyl ketone (zVAD-fmk) and carbobenzyloxy-Ile-Glu(OMe)-Thr-Asp (OMe) fluoromethyl ketone (zIETD-fmk). When used in solution, one of the anti-TRAIL-R2 mAbs was capable of blocking leucine zipper-human TRAIL binding to TRAIL-R2-expressing cells and prevented TRAIL-induced death of these cells, whereas two of the anti-TRAIL-R1 mAbs could inhibit leucine zipper-human TRAIL binding to TRAIL-R1:Fc. Furthermore, use of the blocking anti-TRAIL-R2 mAb allowed us to demonstrate that the signals transduced through either TRAIL-R1 or TRAIL-R2 were necessary and sufficient to mediate cell death. In contrast, the expression of TRAIL-R3 or TRAIL-R4 did not appear to be a significant factor in determining the resistance or sensitivity of these tumor target cells to the effects of TRAIL.
Subject(s)
Antibodies, Monoclonal/immunology , Apoptosis , Receptors, Tumor Necrosis Factor/physiology , Animals , Apoptosis Regulatory Proteins , Caspase Inhibitors , GPI-Linked Proteins , Humans , Membrane Glycoproteins/antagonists & inhibitors , Mice , Mice, Inbred BALB C , Receptors, TNF-Related Apoptosis-Inducing Ligand , Receptors, Tumor Necrosis Factor, Member 10c , TNF-Related Apoptosis-Inducing Ligand , Tumor Cells, Cultured , Tumor Necrosis Factor Decoy Receptors , Tumor Necrosis Factor-alpha/antagonists & inhibitorsABSTRACT
To evaluate the utility of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a cancer therapeutic, we created leucine zipper (LZ) forms of human (hu) and murine (mu) TRAIL to promote and stabilize the formation of trimers. Both were biologically active, inducing apoptosis of both human and murine target cells in vitro with similar specific activities. In contrast to the fulminant hepatotoxicity of LZ-huCD95L in vivo, administration of either LZ-huTRAIL or LZ-muTRAIL did not seem toxic to normal tissues of mice. Finally, repeated treatments with LZ-huTRAIL actively suppressed growth of the TRAIL-sensitive human mammary adenocarcinoma cell line MDA-231 in CB.17 (SCID) mice, and histologic examination of tumors from SCID mice treated with LZ-huTRAIL demonstrated clear areas of apoptotic necrosis within 9-12 hours of injection.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Membrane Glycoproteins/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Animals , Apoptosis Regulatory Proteins , Dose-Response Relationship, Drug , Fas Ligand Protein , Humans , Membrane Glycoproteins/administration & dosage , Membrane Glycoproteins/chemical synthesis , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, SCID , Protein Conformation , TNF-Related Apoptosis-Inducing Ligand , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/administration & dosage , Tumor Necrosis Factor-alpha/chemical synthesisABSTRACT
Staphylococcal superantigens, including staphylococcal enterotoxin B (SEB), promote vigorous T cell-dependent Ig responses at low dose (0.01 ng/ml). In contrast, more mitogenic high dose SEB (100 ng/ml) profoundly inhibits the Ig responses. To assess the contribution of CD8+ T cells to this inhibition, high dose SEB-dependent killing of activated B cells and down-regulation of Ig responses were determined. Rapid killing (4 h) of activated B cells was effected by high dose SEB-activated CD8+ T cells (CD8*), but not by high-dose SEB-activated CD4+ T cells (CD4*), and required the presence of high dose SEB during the cytotoxicity assay. This killing was abrogated by chelation of extracellular calcium or by treatment with concanamycin A but was only modestly affected by treatment with brefeldin A, suggesting a perforin-based pathway of killing. Despite their widely disparate abilities to rapidly kill activated B cells, CD8* and CD4* demonstrated similar quantitative abilities to effect high dose SEB-dependent down-regulation of Ig responses. Antagonist anti-CD95 mAb substantially reversed high dose SEB-dependent downregulation effected by CD8* but had no appreciable effects on high dose SEB-dependent killing of activated B cells. These observations strongly suggest that the small fraction of activated B cells that secrete Ig are selectively sensitive to CD95-based killing but resistant to CD95-independent killing. This finding may help explain why clinical autoimmunity associated with increased titers of autoantibodies is a predominant feature of defects in CD95 or CD95 ligand.
Subject(s)
B-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cytotoxicity, Immunologic , Down-Regulation/immunology , Immunoglobulins/biosynthesis , Lymphocyte Activation , Macrolides , Superantigens/pharmacology , fas Receptor/physiology , Anti-Bacterial Agents/pharmacology , Antibodies, Blocking/pharmacology , Antibodies, Monoclonal/pharmacology , Antibody-Producing Cells/immunology , B-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , Calcium/physiology , Cell Death/drug effects , Cell Death/immunology , Cytotoxicity, Immunologic/drug effects , Cytotoxicity, Immunologic/immunology , Dose-Response Relationship, Immunologic , Enterotoxins/immunology , Enterotoxins/pharmacology , Humans , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Staphylococcus aureus/immunology , Superantigens/immunology , fas Receptor/immunologyABSTRACT
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.