Mitochondrial calcium overload triggers complement-dependent superoxide-mediated programmed cell death in Trypanosoma cruzi

2009 ◽  
Vol 418 (3) ◽  
pp. 595-604 ◽  
Author(s):  
Florencia Irigoín ◽  
Natalia M. Inada ◽  
Mariana P. Fernandes ◽  
Lucía Piacenza ◽  
Fernanda R. Gadelha ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Trypanosoma Cruzi ◽  
Complement Activation ◽  
Superoxide Radical ◽  
Membrane Attack Complex ◽  
Cell Respiration ◽  
Mitochondrial Uncoupling ◽  
O2 Production ◽  
Complement Deposition

The epimastigote stage of Trypanosoma cruzi undergoes PCD (programmed cell death) when exposed to FHS (fresh human serum). Although it has been known for over 30 years that complement is responsible for FHS-induced death, the link between complement activation and triggering of PCD has not been established. We have previously shown that the mitochondrion participates in the orchestration of PCD in this model. Several changes in mitochondrial function were described, and in particular it was shown that mitochondrion-derived O2•− (superoxide radical) is necessary for PCD. In the present study, we establish mitochondrial Ca2+ overload as the link between complement deposition and the observed changes in mitochondrial physiology and the triggering of PCD. We show that complement activation ends with the assembly of the MAC (membrane attack complex), which allows influx of Ca2+ and release of respiratory substrates to the medium. Direct consequences of these events are accumulation of Ca2+ in the mitochondrion and decrease in cell respiration. Mitochondrial Ca2+ causes partial dissipation of the inner membrane potential and consequent mitochondrial uncoupling. Moreover, we provide evidence that mitochondrial Ca2+ overload is responsible for the increased O2•− production, and that if cytosolic Ca2+ rise is not accompanied by the accumulation of the cation in the mitochondrion and consequent production of O2•−, epimastigotes die by necrosis instead of PCD. Thus our results suggest a model in which MAC assembly on the parasite surface allows Ca2+ entry and its accumulation in the mitochondrion, leading to O2•− production, which in turn constitutes a PCD signal.

scholarly journals Cell Death and Metabolic Stress in Gymnodinium catenatum Induced by Allelopathy

Toxins ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 506
Author(s):  
Leyberth José Fernández-Herrera ◽  
Christine Johanna Band-Schmidt ◽  
Tania Zenteno-Savín ◽  
Ignacio Leyva-Valencia ◽  
Claudia Judith Hernández-Guerrero ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Protein Content ◽  
Caspase 3 ◽  
Cellular Stress ◽  
Allelopathic Effect ◽  
Sod Activity ◽  
Free Media ◽  
O2 Production ◽  
Gymnodinium Catenatum

Allelopathy between phytoplankton species can promote cellular stress and programmed cell death (PCD). The raphidophyte Chattonella marina var. marina, and the dinoflagellates Margalefidinium polykrikoides and Gymnodinium impudicum have allelopathic effects on Gymnodinium catenatum; however, the physiological mechanisms are unknown. We evaluated whether the allelopathic effect promotes cellular stress and activates PCD in G. catenatum. Cultures of G. catenatum were exposed to cell-free media of C. marina var. marina, M. polykrikoides and G. impudicum. The mortality, superoxide radical (O2●−) production, thiobarbituric acid reactive substances (TBARS) levels, superoxide dismutase (SOD) activity, protein content, and caspase-3 activity were quantified. Mortality (between 57 and 79%) was registered in G. catenatum after exposure to cell-free media of the three species. The maximal O2●− production occurred with C. marina var. marina cell-free media. The highest TBARS levels and SOD activity in G. catenatum were recorded with cell-free media from G. impudicum. The highest protein content was recorded with cell-free media from M. polykrikoides. All cell-free media caused an increase in the activity of caspase-3. These results indicate that the allelopathic effect in G. catenatum promotes cell stress and caspase-3 activation, as a signal for the induction of programmed cell death.

Metacaspases of Trypanosoma cruzi: Possible candidates for programmed cell death mediators

2006 ◽  
Vol 145 (1) ◽  
pp. 18-28 ◽  
Author(s):  
Gregor Kosec ◽  
Vanina E. Alvarez ◽  
Fernán Agüero ◽  
Daniel Sánchez ◽  
Marko Dolinar ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Trypanosoma Cruzi

Staging of astrocytopathy and complement activation in neuromyelitis optica spectrum disorders

Brain ◽  
2021 ◽  
Author(s):  
Yoshiki Takai ◽  
Tatsuro Misu ◽  
Hiroyoshi Suzuki ◽  
Toshiyuki Takahashi ◽  
Hiromi Okada ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Neuromyelitis Optica ◽  
Complement Activation ◽  
Inflammatory Cell ◽  
Tissue Injury ◽  
Membrane Attack Complex ◽  
Inflammatory Cell Infiltration ◽  
Cell Infiltration ◽  
Polymorphonuclear Cells ◽  
Complement Deposition

Abstract Aquaporin 4 (AQP4)-IgG-positive neuromyelitis optica spectrum disorder (AQP4-IgG+NMOSD) is an autoimmune astrocytopathic disease pathologically characterized by the massive destruction and regeneration of astrocytes with diverse types of tissue injury with or without complement deposition. However, it is unknown whether this diversity is derived from differences in pathological processes or temporal changes. Furthermore, unlike for the demyelinating lesions in multiple sclerosis, there has been no staging of astrocytopathy in AQP4-IgG+NMOSD based on astrocyte morphology. Therefore, we classified astrocytopathy of the disease by comparing the characteristic features, such as AQP4 loss, inflammatory cell infiltration, complement deposition and demyelination activity, with the clinical phase. We performed histopathological analyses in eight autopsied cases of AQP4-IgG+NMOSD. There were six women and two men, with a median age of 56.5 years (range, 46–71 years) and a median disease duration of 62.5 months (range, 0.6–252 months). Astrocytopathy in AQP4-IgG+NMOSD was classified into the following four stages defined by the astrocyte morphology and immunoreactivity for glial fibrillary acidic protein (GFAP): (a) astrocyte lysis: Extensive loss of astrocytes with fragmented and/or dust-like particles; (b) progenitor recruitment: Loss of astrocytes except small nucleated cells with GFAP-positive fibre-forming foot processes; (c) protoplasmic gliosis: Presence of star-shaped astrocytes with abundant GFAP-reactive cytoplasm; and (d) fibrous gliosis: Lesions composed of densely packed mature astrocytes. The astrocyte lysis and progenitor recruitment stages dominated in clinically acute cases (within 2 months after the last recurrence). Findings common to both stages were the loss of AQP4, a decreased number of oligodendrocytes, the selective loss of myelin-associated glycoprotein and active demyelination with phagocytic macrophages. The infiltration of polymorphonuclear cells and T cells (CD4-dominant) and the deposition of activated complement (C9neo), which reflects the membrane attack complex, a hallmark of acute NMOSD lesions, were selectively observed in the astrocyte lysis stage (98.4% in astrocyte lysis, 1.6% in progenitor recruitment, and 0% in protoplasmic gliosis and fibrous gliosis). Although most of the protoplasmic gliosis and fibrous gliosis lesions were accompanied by inactive demyelinated lesions with a low amount of inflammatory cell infiltration, the deposition of complement degradation product (C3d) was observed in all four stages, even in fibrous gliosis lesions, suggesting the past or chronic occurrence of complement activation, which is a useful finding to distinguish chronic lesions in NMOSD from those in multiple sclerosis. Our staging of astrocytopathy is expected to be useful for understanding the unique temporal pathology of AQP4-IgG+NMOSD.

Spinning Disk Confocal Fluorescent Microscopy (SDCFM) Analyses of Complement Activation Promoted by Anti-CD20 Monoclonal Antibodies (mAbs) Rituximab and Ofatumumab.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2345-2345
Author(s):  
Ronald P. Taylor ◽  
Paul V. Beum ◽  
Andrew W. Pawluczkowycz ◽  
Margaret A. Lindorfer ◽  
Frank Beurskens ◽  
...  
Keyword(s):  
Cell Death ◽  
Real Time ◽  
Complement Activation ◽  
Fluorescent Microscopy ◽  
Membrane Attack Complex ◽  
Time Dynamics ◽  
Real Time Analysis ◽  
Centrifugation Step ◽  
Normal Human ◽  
Anti Cd20

Abstract We examined the real-time dynamics of complement activation on CD20-positive cells, promoted by binding of two different anti-CD20 mAbs, rituximab and ofatumumab. Daudi and ARH77 cells were opsonized with Alexa-labeled anti-CD20 mAbs, mixed with normal human serum (NHS) and immediately analyzed by SDCFM at 37°C. Multiple fluorescent image Z stacks were captured over 2–10 min, and Quicktime™ movies generated. Deposition of C3b fragments was monitored with Alexa-labeled mAb 3E7 in situ, specific for cell-bound C3b/iC3b. Complement activation mediated by the mAbs induced profound effects on the cells. C3b deposition was readily demonstrable and substantial changes in morphology including rapid blebbing were evident. Most strikingly, long string-like structures were cast off the cells. These mAb-induced streamers were seen in both NHS and in citrated plasma, but were not generated if complement-mediated killing was precluded, e.g. in C5-deficient NHS or in NHS-EDTA. The streamers appear to be quite fragile and were not observed if cells were subjected to a centrifugation step. In 2-color and 3-color experiments using propidium iodide (PI), cell death was easily seen as PI entered the cells. The streamers also stained positive with phalloidin, and streaming was largely abrogated with cytochalasin D, suggesting that the streamers contained polymerized actin. Movies made under white light conditions also revealed streamers, suggesting that cell-derived fragments, most likely originating at the cell membrane, were being extruded as a consequence of penetration of the cells by the pore-forming membrane attack complex (MAC) of complement. Although not all cells that were killed also produced streamers, the production of streamers, presumably indicative of attack by the MAC, tended to correlate with subsequent killing. In fact, another reagent known to lyse cells based on insertion of a different pore-forming complex, melittin, also induced streamers in these cells. Direct comparisons between rituximab and ofatumumab revealed several interesting differences. Ofatumumab readily promoted complement activation, C3b deposition, and killing of ARH77 cells, but rituximab-mediated C3b deposition was lower, and killing was close to background. In addition, maximum deposition of C3b fragments on ARH77 cells occurred considerably more rapidly for ofatumumab (∼ 30 sec) than for rituximab (∼ 5 min). Consistent with these findings, binding of ofatumumab to ARH 77 cells in NHS induced blebbing and generated streamers to a much greater degree (5 to 10-fold more) than seen for cells opsonized with rituximab and NHS. Moreover, for Daudi cells that were opsonized with the mAbs on ice and then reacted with NHS and placed at 37°C, ofatumumab induced streaming in ∼ 2 min, but rituximab-mediated streaming was not evident until at least 10 min. In summary, SDCFM allows for real time analysis of several distinct steps in mAb-mediated complement activation and killing of targeted cells. Our results indicate that binding of ofatumumab to CD20-positive cells rapidly activates complement and produces profound changes in the cells, including the generation of streamers followed by cell death, in periods of 5 minutes or less.

Complement Deposition and Cell Death after Myoblast Transplantation

1998 ◽  
Vol 7 (5) ◽  
pp. 427-434 ◽  
Author(s):  
Daniel Skuk ◽  
Jacques P. Tremblay
Keyword(s):  
Cell Death ◽  
Intracellular Calcium ◽  
Complement Activation ◽  
Early Death ◽  
Cobra Venom ◽  
Cobra Venom Factor ◽  
Cd1 Mice ◽  
Myoblast Transplantation ◽  
Complement Deposition

One of the problems limiting myoblast transplantation (MT) is the early death of the transplanted cells. Because complement can be fixed by myoblasts in vitro, and because it has the capacity to induce cell lysis, its possible role in the early death of transplanted myoblasts was investigated. CD1 mice and Macaca mulata monkeys were used as recipients for MT. In some mice, C3 was depleted before MT using Cobra Venom Factor. Mice were sacrificed during the first hour and up to 3 days after MT. Monkeys were biopsied 1 to 4 h after MT. Myoblast necrosis was assessed by the presence of intracellular calcium. Complement deposition was demonstrated by immunohistochemistry with anti-C3 and anti-C5b-9 neoantigen antibodies. In mice, C3 deposition was observed in damaged muscle fibers and in regions containing necrosed myoblasts. Complement depletion did not diminish the proportion of necrosed cells. In monkeys, only a small percentage of transplanted myoblasts showed C3 or C5b-9 deposition, mostly intracellular. Complement activation seems not to be implicated in directly damaging the transplanted cells, but seems secondary to cellular death. Taking into account its chemotactic functions, complement could be implicated in the migration of neutrophils and macrophages into the clusters of transplanted cells. © 1998 Elsevier Science Inc.

scholarly journals Mitochondrial superoxide radicals mediate programmed cell death in Trypanosoma cruzi: cytoprotective action of mitochondrial iron superoxide dismutase overexpression

2007 ◽  
Vol 403 (2) ◽  
pp. 323-334 ◽  
Author(s):  
Lucía Piacenza ◽  
Florencia Irigoín ◽  
María Noel Alvarez ◽  
Gonzalo Peluffo ◽  
Martin C. Taylor ◽  
...  
Keyword(s):  
Cell Death ◽  
Superoxide Dismutase ◽  
Programmed Cell Death ◽  
Trypanosoma Cruzi ◽  
Early Period ◽  
Pentose Phosphate ◽  
Iron Superoxide Dismutase ◽  
Mitochondrial Superoxide ◽  
Glucose Flux ◽  
Mitochondrial Aconitase

Trypanosoma cruzi undergo PCD (programmed cell death) under appropriate stimuli, the mechanisms of which remain to be established. In the present study, we show that stimulation of PCD in T. cruzi epimastigotes by FHS (fresh human serum) results in rapid (<1 h) externalization of phosphatidylserine and depletion of the low molecular mass thiols dihydrotrypanothione and glutathione. Concomitantly, enhanced generation of oxidants was established by EPR and immuno-spin trapping of radicals using DMPO (5,5-dimethylpyrroline-N-oxide) and augmentation of the glucose flux through the pentose phosphate pathway. In the early period (<20 min), changes in mitochondrial membrane potential and inhibition of respiration, probably due to the impairment of ADP/ATP exchange with the cytosol, were observed, conditions that favour the generation of O2•−. Accelerated rates of mitochondrial O2•− production were detected by the inactivation of the redox-sensitive mitochondrial aconitase and by oxidation of a mitochondrial-targeted probe (MitoSOX). Importantly, parasites overexpressing mitochondrial FeSOD (iron superoxide dismutase) were more resistant to the PCD stimulus, unambiguously indicating the participation of mitochondrial O2•− in the signalling process. In summary, FHS-induced PCD in T. cruzi involves mitochondrial dysfunction that causes enhanced O2•− formation, which leads to cellular oxidative stress conditions that trigger the initiation of PCD cascades; moreover, overexpression of mitochondrial FeSOD, which is also observed during metacyclogenesis, resulted in cytoprotective effects.

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