Roles of ICAM-3 and CD14 in the recognition and phagocytosis of apoptotic cells by macrophages.
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Animals↗
Biomedical subjects
C D Gregory
Publications and source records attributed to C D Gregory.
Mechanisms of antigen receptor-dependent apoptosis of human B lymphoma cells probed with a panel of 27 monoclonal antibodies.
The present study has used a panel of 23 monoclonal antibodies to IgM and 4 to IgD in order to probe parameters influencing sIg-dependent apoptosis in an IgM/IgD-expressing Burkitt lymphoma line. No direct correlation was observed between the capacity of the different anti-mu to drive cells into apoptosis and either their domain specificity or their affinity for sIgM. There was, however, a direct correlation between the functional outcome and the ability of the monoclonal antibodies to elicit a rise in intracellular Ca2+. For apoptosis to occur, the Ca2+ response had to attain a threshold value of approximately 100 nM. A direct role for Ca2+ in the delivery of the apoptotic signal was demonstrated using thapsigargin to raise intracellular Ca2+ levels. Antigen receptor ligation was linked to Ca2+ increases by tyrosine kinases as revealed by direct analysis of protein tyrosine phosphorylation and the effects of selective protein tyrosine kinase-inhibiting tyrphostins. These findings reveal a central role for the antigen receptor-generated Ca2+ signal in driving apoptosis in human B lymphoma cells and stresses the need to use a panel of reagents when probing function with presumed ligand-mimetic monoclonal antibodies.
Antibodies, Monoclonal↗
Differential effects of BCL-2 on survival and proliferation of human B-lymphoma cells following gamma-irradiation.
Bcl-2 can inhibit apoptosis induced by a variety of stimuli, including radiation and its presence in tumour cells would be expected to indicate poor prognosis. Bcl-2-expressing tumours, however, are often low-grade and highly responsive to therapy. To investigate this apparent paradox, we analysed in vitro the responses of Burkitt lymphoma (BL) cells to gamma-irradiation in the presence and absence of Bcl-2. High-level expression of Bcl-2 was shown to promote BL cell survival following irradiation. However, a significant proportion of Bcl-2-rescued cells subsequently underwent apoptosis after an extended period in culture. In addition, in different BL lines, Bcl-2 was found either to promote or to inhibit long-term proliferative activity following gamma-irradiation. This differential regulation of proliferation correlated both with differential effects of Bcl-2 on the cell cycle and with differences in p53 status. Thus, by one week after irradiation, BL cells expressing only wild-type p53 (wt/wt) had arrested in G1, whereas those with a mutant allele (wt/mu) were arrested in all phases of the cell cycle. The proportion of Bcl-2-rescued cells that subsequently underwent apoptosis was reduced by ligation of CD40 at the time of irradiation in wt/wt BL cells, but not in wt/mu cells. CD40-ligation reduced both G1-arrest and apoptosis in parallel. These results indicate that, whilst Bcl-2 can delay apoptosis in BL cells following gamma-irradiation, the protein can also cause growth-arrest and thereby promote apoptosis. Long-term survival following Bcl-2-mediated rescue of gamma-irradiated cells may depend on p53 status and require additional death-repressing or growth-promoting signals.
CD40 Antigens↗
Biochemical heterogeneity in hysterectomized uterus measured by 31P NMR using SLIM localization.
Ultrasound and magnetic resonance imaging show contrast between the inner and outer myometrium, which is useful in the diagnosis of gynecological disorders. To determine whether the image contrast is associated with biochemical differences between these myometrial regions, phosphorus metabolite concentrations in the inner one third of the myometrium (the junctional zone; JZ) were compared with the outermost one third of the myometrium (OM) in hysterectomized uteri using 31P spectral localization by imaging (SLIM). The technique was validated by comparing the results of SLIM with the results of standard Fourier-encoded spectroscopic imaging (FSI) analysis using phantoms, and by nonlocalized spectroscopy on biopsies taken from the same hysterectomy specimens. As expected theoretically, SLIM yielded better localization than FSI, as judged by spectral intensity and leakage measurements on phantom compartments of known composition. SLIM localization revealed that the JZ has a higher intracellular phosphomonoester (PME) concentration than does the OM, which was confirmed by nonlocalized spectroscopy, and that there is very little NMR-visible phosphorus in the cervix.
Adult↗
Quantitative lactate-specific MR imaging and 1H spectroscopy of skeletal muscle at macroscopic and microscopic resolutions using a zero-quantum/double-quantum coherence filter and SLIM/GSLIM localization.
Quantitative lactate imaging and spectroscopy were performed on phantoms and on electrically stimulated, excised frog skeletal muscle at macroscopic and microscopic resolutions. Lactate selectivity was achieved by use of a zero-quantum/double-quantum coherence (ZQC/DQC) lactate filter, which suppressed all signals besides lactate, including water and lipid, to below noise level. Three-dimensional lactate data sets were acquired in 1-3 h; one of these spatial dimensions was frequency-encoded and the other two were phase-encoded. High-resolution images were reconstructed using the spectral localization by imaging (SLIM) and generalized SLIM (GSLIM) techniques. Lactate quantitation was achieved by employing an external lactate concentration standard and was verified by comparison to quantitative STEAM-localized and nonlocalized spectra that used total creatine as an internal concentration reference. Additionally, quantitatively accurate behavior of the SLIM and GSLIM techniques as applied to data sets of low signal-to-noise ratio and to macroscopically heterogeneous objects was verified using simulations and real muscle lactate data sets with known heterogeneity.
Animals↗
Projection-reconstruction spectroscopic imaging for B0 field plotting and shimming without pulsed gradients.
Shimming is important. Noniterative methods are desirable. Such methods exist for shimming a spectrometer with pulsed field gradients, generally based on field maps made by spin-warp Fourier imaging. For spectrometers with no pulsed gradients (or for cases where T2 is too short to permit echo imaging), an alternative method is presented: projection-reconstruction spectroscopic imaging, which can be accomplished using only the shim coils of a conventional spectrometer. Images so acquired can be used to map the field, even in the presence of multiple spectral components. Noniterative optimization of the axial shims of a GN-300 spectrometer is demonstrated using 1D + 1D spectroscopic images. Prospects for extending the technique to include the radial shims using 3D + 1D spectroscopic images are discussed.
Fourier Analysis↗
Repression of apoptosis in human B-lymphoma cells by CD40-ligand and Bcl-2: relationship to the cell-cycle and role of the retinoblastoma protein.
Using a Burkitt lymphoma cell line to model human B-cell apoptosis in vitro, we observed that crosslinking, by antibody, of cell surface immunoglobulin induced G1 growth-arrest followed by apoptosis. By contrast, cells treated with the Ca(2+)-ionophore, ionomycin, generated apoptotic signals in G2/M as well as in G1. Both ionomycin and anti-immunoglobulin treatment induced rapid dephosphorylation of Rb prior to apoptosis. Apoptosis was repressed following exposure to CD40-ligand and was accompanied by hyperphosphorylation of Rb and cell-cycle progression but not Bcl-2 expression. Expression of Bcl-2 protein in stable bcl-2-transfectants, also resulted in repression of apoptosis and anti-immunoglobulin-treated cells no longer underwent growth-arrest. In Bcl-2-expressing cells in which apoptosis was repressed, Rb remained hyperphosphorylated, even during G1-arrest induced by ionomycin. TGF beta treatment of Bcl-2-expressing cells induced G1-arrest, de-phosphorylation of Rb and apoptosis. These results suggest that the functional activity of Bcl-2 in B-lymphoma cells is dependent upon, or leads to, sustained hyperphosphorylation of Rb and that Rb hyperphosphorylation can be uncoupled from cell-cycle progression.
Antibodies, Anti-Idiotypic↗
Transforming growth factor-beta 1 cooperates with anti-immunoglobulin for the induction of apoptosis in group I (biopsy-like) Burkitt lymphoma cell lines.
Group I Burkitt lymphoma (BL) cell lines, which retain the original biopsy phenotype, have been shown to enter apoptosis in response to a number of external stimuli including serum deprivation, thermal shock, addition of calcium ionophore, and ligation of surface immunoglobulin (Ig) by antibody. Transforming growth factor-beta 1 (TGF beta 1) is known to cause growth arrest in BL lines. Here we show that while it is by itself capable of promoting some degree of apoptosis in group IBL cells, TGF beta 1 cooperates with anti-immunoglobulin to this end. Trimeric soluble recombinant human CD40 ligand (sCD40L) was able to inhibit apoptosis induced by the combination of agonists to some degree, but such rescue proved to be short-lived. Both TGF beta 1 and anti-Ig individually caused BL cells to undergo growth arrest at the G1 phase of cell cycle before their entry into apoptosis: the consequence of sCD40L addition was to maintain the cells in cycle for longer. No induction of the apoptosis-protecting gene, bcl-2, occurred in the presence of sCD40L. These findings are discussed, particularly highlighting the relationship existing between survival and the cell cycle. The strong cooperative effects observed between anti-Ig and TGF beta 1 in promoting apoptosis and the inability of CD40 to signal for long-term rescue raise the potential for a novel therapeutic attack on B-cell lymphoma.
Animals↗
Bcl-2 delays macrophage engulfment of human B cells induced to undergo apoptosis.
The capacity to be recognized and engulfed by phagocytes is an important characteristic of cells dying by apoptosis. Phagocytosis of apoptotic cells occurs rapidly in vivo, probably prior to plasma membrane breakdown. While the molecular mechanisms mediating phagocytosis of apoptotic cells are beginning to be defined, little is yet known of the relationship between the cell-death program itself and the surface changes on the dying cells that signal for engulfment. Here, we investigate to what extent the apoptosis repressor Bcl-2 can modulate the recognition and phagocytosis of human B cells exposed to triggers of apoptosis. Burkitt lymphoma (BL)-derived, Bcl-2- B cells were induced into apoptosis either by the Ca(2+)-ionophore ionomycin or by the inhibitor of protein synthesis cycloheximide. Apoptotic BL cells, but not viable BL cells, were recognized and phagocytosed by monocyte-derived macrophages. bcl-2-transfected BL populations showed a reduced capacity both to undergo apoptosis in response to these inducing agents and to interact with macrophages. Like their Bcl-2- counterparts, Bcl-2+ BL cells interacted with macrophages only after activation of their apoptotic program as assessed by changes in nuclear morphology. These results demonstrate not only that continued protein synthesis in B cells undergoing apoptosis is not essential for their recognition by macrophages, but also that macrophage recognition of apoptotic B cells cannot be uncoupled from the cell-death program that is controlled by Bcl-2. In this respect, the behavior of B cells contrasts markedly with that of neutrophils in which Bcl-2 has been reported to inhibit apoptosis without affecting phagocytic clearance.
Apoptosis↗
Observation and quantitation of lactate in oxidative and glycolytic fibers of skeletal muscles.
In 1H NMR spectroscopic studies of fatiguing skeletal muscles, two peaks consistently arise near 1.3 ppm, typically 15-20 Hz apart at 300 MHz. From a variety of NMR and biochemical evidence, both peaks are identified as lactate. Both the CH3 and CH protons of lactate experience the same shift in intact muscle; this rules out chemical bonding or complexation. The ratio of intensity of the two methyl peaks varies with muscle type and suggests a correlation with oxidative and glycolytic fiber populations. The shift can be accounted for by the presence of paramagnetic myoglobin in the oxidative fibers. Phantom studies, as well as oxygen, temperature, field, and orientation dependence of the muscle spectra are all consistent with an explanation based upon bulk magnetic susceptibility. It is concluded that the two lactate peaks represent separate contributions from glycolytic and oxidative muscle fibers.
Animals↗
Signals for survival and apoptosis in normal and neoplastic B lymphocytes.
B lymphocytes are subject to selection within germinal centers following somatic hyper-mutation on immunoglobulin variable region genes based on their ability to bind antigen with high affinity. Non-selected cells die by apoptosis. Tumors with features of germinal center B cells include follicular center cell lymphoma and Burkitt's lymphoma. We have used the latter extensively as a neoplastic model of germinal center cells and have compared directly the behaviour of cell lines derived from biopsy material with that of the normal counterparts. Here we describe some of our findings in the two systems with regard to signals regulating survival and apoptosis.
Animals↗
Effects of interferon-alpha on human B cells: repression of apoptosis and prevention of cell growth are independent responses of Burkitt lymphoma lines.
We have previously shown that interferon-alpha (IFN-alpha) can repress apoptosis in Burkitt lymphoma (BL) cells. In this study, we have compared this protective response with a further, well-established effect of IFN-alpha on BL cells, that of growth arrest. Of a panel of BL lines comprising (i) EBV-positive and -negative lines that retain the phenotype of the parental tumour cells and (ii) the prototype IFN-alpha-growth-inhibited line, Daudi, only Daudi cells were found to undergo substantial growth inhibition in response to the cytokine. By contrast, all lines, with the notable exception of Daudi, were protected by IFN-alpha from high-rate apoptosis initiated by the Ca2+ ionophore ionomycin. Ionomycin failed to elicit an IFN-alpha-repressible apoptotic response in either wild-type Daudi cells or IFN-resistant sublines that were refractory to the growth-arresting effects of the cytokine. Analysis of c-myc protein levels confirmed previous observations that repression of apoptosis in IFN-alpha-rescuable BL cells was associated with an early inhibition of myc that was followed by a return to high-level expression. Significantly, ionomycin alone induced a comparable transient inhibition of myc protein in Daudi cells. In Daudi cells, but not in IFN-alpha-rescuable BL cells, renewed expression of myc observed after the early, transient down-regulation was followed by sustained down-regulation of the protein, which paralleled growth arrest. Our results indicate that long-term growth arrest and repression of apoptosis in BL are distinct cellular responses to IFN-alpha.
Apoptosis↗
A novel protein expressed in mammalian cells undergoing apoptosis.
Human and rodent cells undergoing apoptosis were observed to express high levels of a novel 45,000 M(r) protein. The protein, which we have termed apoptosis specific protein (ASP), was found in Burkitt lymphoma (BL) cells and in adenovirus-transformed human and rat embryo cells induced into apoptosis by a variety of stimuli, including serum deprivation, exposure to the Ca2+ ionophore, ionomycin, treatment with inhibitors of macromolecular synthesis (cycloheximide and actinomycin D), and cold shock. In BL cells treated with apoptotic stimuli, expression of the oncoprotein Bcl-2 was found to both protect from apoptosis and prevent expression of ASP. ASP was not detected either in viable cells or in cells dying passively by necrosis. Laser scanning confocal microscopy showed high levels of ASP in the cytoplasm of cells displaying the chromatin condensation and fragmentation patterns typical of apoptosis. Retention of ASP was observed even when DNA was no longer detectable, and two-color immunofluorescence staining indicated that the protein primarily colocalized with, but was clearly distinct from, non-muscle actin. These findings, together with the observation that biochemical extraction of ASP was only possible under conditions which caused solubilization of the cytoskeleton, leads us to conclude that ASP forms part of, or at least strongly associates with, a modified cytoskeleton unique to cells undergoing apoptosis. While elucidation of its function will require further work, ASP constitutes a powerful marker for the diagnosis and quantitation of apoptosis in vivo and in vitro.
Animals↗
Induction of latent membrane protein expression in in vitro Epstein-Barr virus-infected leukaemic B lymphocytes by interleukin 4 and antibodies to CD40.
Chronic lymphocytic leukaemia (CLL) B cells are clones representing the mature B cell phenotype. On infection with Epstein-Barr virus (EBV) CLL cells express the EB nuclear antigen (EBNA) complex but unlike EBV-infected normal B cells they do not express LMP nor do they proliferate or immortalize. Furthermore, EBV-CLL rapidly die by apoptosis in culture. In the present study we have used the B cell growth factors interleukin 4 and antibodies to CD40 to induce activation and proliferation of EBV-infected CLL cells. Although cell numbers did not significantly increase, apoptosis was partially inhibited in CLL cells which expressed increased levels of CD23 and were activated to immunoglobulin-secreting lymphoblasts. Expression of LMP was induced by interleukin (IL)-4 and anti-CD40 in all five EBV-infected CLL samples examined. However, this did not enhance cell proliferation or induce immortalization. Further analysis showed that LMP could be detected 4-5 days after EBV infection, and that both IL-4 and anti-CD40 could independently induce LMP but that their effect was additive. These results indicate that LMP expression is dependent on B cell activation processes and that in some circumstances full latent viral gene expression is not sufficient to cause B cell immortalization.
Antibodies↗
Regulation of cell survival in Burkitt lymphoma: implications from studies of apoptosis following cold-shock treatment.
Burkitt lymphoma (BL) tumour-cell populations are known to display high rates both of proliferation and of apoptosis in vivo, but the mechanisms which determine whether a BL cell continues to cycle or engages its cell-death programme are not understood. Group-I BL-derived cell lines, which retain in vitro the proliferative and apoptotic capacities of the parental cells, selectively entered apoptosis when returned to 37 degrees C after a brief period at low temperature (1 degree C). The induction of apoptosis by cold treatment, as determined by morphological characteristics and DNA fragmentation, was readily detectable within the first 1 to 2 hr of re-incubation at 37 degrees C, reaching a maximum at 4 to 6 hours. Commitment to enter apoptosis occurred after as little as 20 to 30 min at 1 degree C. Significant cell death at 1 degree C occurred only during prolonged incubation in the cold and displayed the characteristics of necrosis. Both bcl-2-dependent and -independent survival pathways were found to provide protection from cold-induced apoptosis, but only if engaged before cold-shock treatment. These results indicate that continued cycling of group-I BL cells is dependent upon their capacity to inhibit or circumvent their normally constitutively active apoptotic programme, and are consistent with the notion that the synthesis of one or more critical "survival" proteins of short half-life is necessary to guarantee successful passage through the cell cycle. Notably, high levels of apoptosis were also inducible in group-I BL cells by inhibitors of RNA and protein synthesis.
Apoptosis↗
Recognition of apoptotic cells by human macrophages: inhibition by a monocyte/macrophage-specific monoclonal antibody.
Cells undergoing death by apoptosis are rapidly engulfed by phagocytes in vivo, a highly efficient process which prevents leakage of potentially dangerous intracellular contents from dying cells to neighboring tissue. We have tested a panel of monoclonal antibodies (mAb) specifying a range of human monocyte/macrophage surface antigens for their capacity to inhibit the in vitro recognition of apoptotic cells by human peripheral blood monocyte-derived macrophages. The results identify the antigen defined by the 61D3 mAb, a widely-used marker of monocyte/macrophage lineage cells, as an important mediator of apoptotic cell recognition. In our system, apoptotic, but not viable, cells were recognized by the cultured macrophages and 61D3 was found to inhibit the recognition of all apoptotic cell types tested, including Ca2+ ionophore-treated or growth factor-depleted B and T lymphocyte lines, tonsillar germinal center B cells, irradiated peripheral blood lymphocytes and senescing neutrophils. Furthermore, the apoptotic cell recognition pathway specified by 61D3 could be distinguished from that involving the macrophage alpha v beta 3 vitronectin receptor which has been shown previously to play an important role in the recognition of apoptotic cells. These results provide further evidence that the mechanisms underlying rapid clearance of apoptotic cells involve multiple phagocyte receptors.
Animals↗
Irradiated fibroblasts protect Burkitt lymphoma cells from apoptosis by a mechanism independent of bcl-2.
When cells of fresh Burkitt lymphoma (BL) biopsies are explanted into tissue culture, their survival and growth are greatly dependent on the presence of a feeder layer of irradiated fibroblasts. To investigate the nature of this feeder dependence, we characterized the growth requirements of a panel of Epstein-Barr Virus (EBV)-negative and -positive BL cell lines in both the absence and the presence of feeder cells in vitro. Four EBV-negative BL lines and 4 EBV-positive lines displaying the phenotype of BL cells in vivo required high cell density for proliferation in the absence of feeder cells, but grew out as single-cell clones when seeded on irradiated human fibroblasts. EBV-positive BL cell lines which had acquired an activated phenotype similar to that of lymphoblastoid cell lines required much lower cell densities for autonomous proliferation. The EBV-negative Burkitt lymphoma line BL70 was used as a model to study the feeder-cell dependence in more detail. BL70 cells grew in the absence of feeder cells only at high cell density and at high FCS concentration. In the presence of feeder cells, BL70 cells became clonogenic even at greatly reduced FCS concentration. A decrease in either cell density or FCS concentration induced apoptosis. Supernatants from feeder cells and from BL70 cells growing autonomously at high cell density were unable to substitute for the survival and growth-promoting effect of the feeder cells. Protection of Burkitt lymphoma cells from apoptosis by co-cultivation with irradiated fibroblasts was not mediated by induction of bcl-2.(ABSTRACT TRUNCATED AT 250 WORDS)
Apoptosis↗
bcl-2 modulation of apoptosis induced by anticancer drugs: resistance to thymidylate stress is independent of classical resistance pathways.
The hypothesis was tested that expression of bcl-2 could provide protection against apoptosis induced by cytotoxic drugs via a mechanism which was different from the classical determinants of drug resistance. Sensitivity and resistance to inhibitors of thymidylate synthase (EC 2.1.1. 45) were chosen for study since these drugs have a well-defined and quantifiable locus of action with similarly well defined biochemical sequelae resulting from enzyme inhibition. Human lymphoma cells transfected with the vector alone readily underwent apoptosis after a 36-h exposure to various drugs. For example, 5-fluorodeoxyuridine (0.1 microM) induced 67% apoptosis in vector control cells 24 h after removal of the drug. In contrast, cells treated under identical conditions, but which expressed the bcl-2 protein, showed only basal levels of apoptosis (8%), with no significant fall in viability. Similar results were obtained using two quinazoline-based inhibitors of thymidylate synthase, N10-propargyl-5,8-dideazafolic acid (CB3717) and ICI M247496. Determinants of resistance to these three drugs were investigated. Analysis of the cell cycle, thymidylate synthase levels, and activity showed these to be unchanged by expression of bcl-2. Addition of the drugs brought about equivalent inhibition of proliferation in the presence or absence of bcl-2 expression. 5-Fluorodeoxyuridine treatment reduced TTP synthesis, induced strand breaks in nascent DNA, measured by alkaline elution, and increased the synthesis of thymidylate synthase; these changes preceded the onset of apoptosis and were identical in the vector controls and bcl-2 transfectants. Resistance to thymidylate stress in bcl-2-expressing cells therefore occurred by a mechanism different from those which classically define resistance to this type of cytotoxic drug.
Apoptosis↗