The autoimmune conundrum in common variable immunodeficiency disorders.

PURPOSE OF REVIEW: Autoimmune and inflammatory manifestations are the biggest clinical challenge in the care of patients with common variable immunodeficiency (CVID). The increasing pathogenic knowledge and potential therapeutic implications require a new evaluation of the status quo. (Figure is included in full-text article.) RECENT FINDINGS: The conundrum of the simultaneous manifestation of primary immunodeficiency and autoimmune disease (AID) is increasingly elucidated by newly discovered genetic defects. Thus, cytotoxic T lymphocyte-associated antigen 4 or caspase-9 deficiency presenting with CVID-like phenotypes reiterate concepts of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome and autoimmune lymphoproliferative syndrome. Activating signaling defects downstream of antigen or cytokine receptors are often associated with loss-of-tolerance in the affected patients. Increasingly, forms of combined immunodeficiency are discovered among CVID-like patients. Although different autoimmune manifestations often coincide in the same patient their immunopathology varies. Treatment of AID in CVID remains a challenge, but based on a better definition of the immunopathology first attempts of targeted treatment have been made. SUMMARY: The increasing comprehension of immunological concepts promoting AID in CVID will allow better and in some cases possibly even targeted treatment. A genetic diagnosis therefore becomes important information in this group of patients, especially in light of the fact that some patients might require hematopoietic stem cell transplantation because of their underlying immunodeficiency.

TNFα-induced lysosomal membrane permeability is downstream of MOMP and triggered by caspase-mediated NDUFS1 cleavage and ROS formation.

When NF-κB activation or protein synthesis is inhibited, tumor necrosis factor alpha (TNFα) can induce apoptosis through Bax- and Bak-mediated mitochondrial outer membrane permeabilization (MOMP) leading to caspase-3 activation. Additionally, previous studies have implicated lysosomal membrane permeability (LMP) and formation of reactive oxygen species (ROS) as early steps of TNFα-induced apoptosis. However, how these two events connect to MOMP and caspase-3 activation has been largely debated. Here, we present the novel finding that LMP induced by the addition of TNFα plus cycloheximide (CHX), the release of lysosomal cathepsins and ROS formation do not occur upstream but downstream of MOMP and require the caspase-3-mediated cleavage of the p75 NDUFS1 subunit of respiratory complex I. Both a caspase non-cleavable p75 mutant and the mitochondrially localized antioxidant MitoQ prevent LMP mediated by TNFα plus CHX and partially interfere with apoptosis induction. Moreover, LMP is completely blocked in cells deficient in both Bax and Bak, Apaf-1, caspase-9 or both caspase-3 and -7. Thus, after MOMP, active caspase-3 exerts a feedback action on complex I to produce ROS. ROS then provoke LMP, cathepsin release and further caspase activation to amplify TNFα apoptosis signaling.

Zinc oxide nanoparticles induce necrosis and apoptosis in macrophages in a p47phox- and Nrf2-independent manner.

In view of the steadily increasing use of zinc oxide nanoparticles in various industrial and consumer applications, toxicological investigations to evaluate their safety are highly justified. We have investigated mechanisms of ZnO nanoparticle-induced apoptosis and necrosis in macrophages in relation to their important role in the clearance of inhaled particulates and the regulation of immune responses during inflammation. In the murine macrophage RAW 264.7 cell line, ZnO treatment caused a rapid induction of nuclear condensation, DNA fragmentation, and the formation of hypodiploid DNA nuclei and apoptotic bodies. The involvement of the essential effector caspase-3 in ZnO-mediated apoptosis could be demonstrated by immunocytochemical detection of activated caspase-3 in RAW 264.7 cells. ZnO specifically triggered the intrinsic apoptotic pathway, because Jurkat T lymphocytes deficient in the key mediator caspase-9 were protected against ZnO-mediated toxicity whereas reconstituted cells were not. ZnO also caused DNA strand breakage and oxidative DNA damage in the RAW 264.7 cells as well as p47(phox) NADPH oxidase-dependent superoxide generation in bone marrow-derived macrophages. However, ZnO-induced cell death was not affected in bone marrow-derived macrophages of mice deficient in p47(phox) or the oxidant responsive transcription factor Nrf2. Taken together, our data demonstrate that ZnO nanoparticles trigger p47(phox) NADPH oxidase-mediated ROS formation in macrophages, but that this is dispensable for caspase-9/3-mediated apoptosis. Execution of apoptotic cell death by ZnO nanoparticles appears to be NADPH oxidase and Nrf2-independent but rather triggered by alternative routes.

Caspase-9 mediates the apoptotic death of megakaryocytes and platelets, but is dispensable for their generation and function.

Apoptotic caspases, including caspase-9, are thought to facilitate platelet shedding by megakaryocytes. They are known to be activated during platelet apoptosis, and have also been implicated in platelet hemostatic responses. However, the precise requirement for, and the regulation of, apoptotic caspases have never been defined in either megakaryocytes or platelets. To establish the role of caspases in platelet production and function, we generated mice lacking caspase-9 in their hematopoietic system. We demonstrate that both megakaryocytes and platelets possess a functional apoptotic caspase cascade downstream of Bcl-2 family-mediated mitochondrial damage. Caspase-9 is the initiator caspase, and its loss blocks effector caspase activation. Surprisingly, steady-state thrombopoiesis is unperturbed in the absence of caspase-9, indicating that the apoptotic caspase cascade is not required for platelet production. In platelets, loss of caspase-9 confers resistance to the BH3 mimetic ABT-737, blocking phosphatidylserine (PS) exposure and delaying ABT-737-induced thrombocytopenia in vivo. Despite this, steady-state platelet lifespan is normal. Casp9(-/-) platelets are fully capable of physiologic hemostatic responses and functional regulation of adhesive integrins in response to agonist. These studies demonstrate that the apoptotic caspase cascade is required for the efficient death of megakaryocytes and platelets, but is dispensable for their generation and function.

Lidocaine induces apoptosis via the mitochondrial pathway independently of death receptor signaling.

BACKGROUND: Local anesthetics, especially lidocaine, can lead to persistent cauda equina syndrome after spinal anesthesia. Recently, lidocaine has been reported to trigger apoptosis, although the underlying mechanisms remain unknown. To elucidate the pathway of lidocaine-induced apoptosis, the authors used genetically modified cells with overexpression or deficiencies of key regulators of apoptosis. METHODS: Human Jurkat T-lymphoma cells overexpressing the antiapoptotic protein B-cell lymphoma 2 as well as cells deficient of caspase 9, caspase 8, or Fas-associated protein with death domain were exposed to lidocaine and compared with parental cells. The authors evaluated cell viability, mitochondrial alterations, cytochrome c release, caspase activation, and early apoptosis. Apoptosis was in addition investigated in neuroblastoma cells. RESULTS: In Jurkat cells, lidocaine reduced viability, associated with a loss of the mitochondrial membrane potential. At low concentrations (3-6 mm) of lidocaine, caspase 3 was activated and release of cytochrome c was detected, whereas at higher concentrations (10 mm), no caspase activation was found. Apoptosis by lidocaine was strongly reduced by B-cell lymphoma-2 protein overexpression or caspase-9 deficiency, whereas cells lacking the death receptor pathway components caspase 8 and Fas-associated protein with death domain were not protected and displayed similar apoptotic alterations as the parental cells. Lidocaine also induced apoptotic caspase activation in neuroblastoma cells. CONCLUSIONS: Apoptosis is triggered by concentrations of lidocaine occurring intrathecally after spinal anesthesia, whereas higher concentrations induce necrosis. The data indicate that death receptors are not involved in lidocaine-induced apoptosis. In contrast, the observation that B-cell lymphoma-2 protein overexpression or the lack of caspase 9 abolished apoptosis clearly implicates the intrinsic mitochondrial death pathway in lidocaine-induced apoptosis.

Loss of Mcl-1 protein and inhibition of electron transport chain together induce anoxic cell death.

How cells die in the absence of oxygen (anoxia) is not understood. Here we report that cells deficient in Bax and Bak or caspase-9 do not undergo anoxia-induced cell death. However, the caspase-9 null cells do not survive reoxygenation due to the generation of mitochondrial reactive oxygen species. The individual loss of Bim, Bid, Puma, Noxa, Bad, caspase-2, or hypoxia-inducible factor 1beta, which are potential upstream regulators of Bax or Bak, did not prevent anoxia-induced cell death. Anoxia triggered the loss of the Mcl-1 protein upstream of Bax/Bak activation. Cells containing a mitochondrial DNA cytochrome b 4-base-pair deletion ([rho(-)] cells) and cells depleted of their entire mitochondrial DNA ([rho(0)] cells) are oxidative phosphorylation incompetent and displayed loss of the Mcl-1 protein under anoxia. [rho(0)] cells, in contrast to [rho(-)] cells, did not die under anoxia. However, [rho(0)] cells did undergo cell death in the presence of the Bad BH3 peptide, an inhibitor of Bcl-X(L)/Bcl-2 proteins. These results indicate that [rho(0)] cells survive under anoxia despite the loss of Mcl-1 protein due to residual prosurvival activity of the Bcl-X(L)/Bcl-2 proteins. Collectively, these results demonstrate that anoxia-induced cell death requires the loss of Mcl-1 protein and inhibition of the electron transport chain to negate Bcl-X(L)/Bcl-2 proteins.

Primary enamel knot cell death in Apaf-1 and caspase-9 deficient mice.

During molar development, apoptosis occurs in a well-characterised pattern suggesting several roles for cell death in odontogenesis. However, molecular mechanisms of dental apoptosis are only poorly understood. In this study, Apaf-1 and caspase-9 knockouts were used to uncover the engagement of these members of the apoptotic machinery during early tooth development, concentrating primarily on their function in the apoptotic elimination of primary enamel knot cells. Molar tooth germ morphology, proliferation and apoptosis were investigated on frontal histological sections of murine heads at embryonic days (ED) 15.5, the stage when the primary enamel knot is eliminated apoptotically. In molar tooth germs of both knockouts, no apoptosis was observed according to morphological (haematoxylin-eosin) as well as biochemical criteria (TUNEL). Morphology of the mutant tooth germs, however, was not changed. Additionally, knockout mice showed no changes in proliferation compared to wild type mice. According to our findings on knockout embryos, Apaf-1 and caspase-9 are involved in apoptosis during tooth development; however, they seem dispensable and not necessary for proper tooth shaping. Compensatory or other mechanisms of cell death may act to eliminate the primary enamel knot cells in the absence of Apaf-1 and caspase-9.

Loss of caspase-9 reveals its essential role for caspase-2 activation and mitochondrial membrane depolarization.

Caspase-9 plays an important role in apoptosis induced by genotoxic stress. Irradiation and anticancer drugs trigger mitochondrial outer membrane permeabilization, resulting in cytochrome c release and caspase-9 activation. Two highly contentious issues, however, remain: It is unclear whether the loss of the mitochondrial membrane potential DeltaPsi(M) contributes to cytochrome c release and whether caspases are involved. Moreover, an unresolved question is whether caspase-2 functions as an initiator in genotoxic stress-induced apoptosis. In the present study, we have identified a mutant Jurkat T-cell line that is deficient in caspase-9 and resistant to apoptosis. Anticancer drugs, however, could activate proapoptotic Bcl-2 proteins and cytochrome c release, similarly as in caspase-9-proficient cells. Interestingly, despite these alterations, the cells retained DeltaPsi(M). Furthermore, processing and enzyme activity of caspase-2 were not observed in the absence of caspase-9. Reconstitution of caspase-9 expression restored not only apoptosis but also the loss of DeltaPsi(M) and caspase-2 activity. Thus, we provide genetic evidence that caspase-9 is indispensable for drug-induced apoptosis in cancer cells. Moreover, loss of DeltaPsi(M) can be functionally separated from cytochrome c release. Caspase-9 is not only required for DeltaPsi(M) loss but also for caspase-2 activation, suggesting that these two events are downstream of the apoptosome.

Loss of caspase-9 provides genetic evidence for the type I/II concept of CD95-mediated apoptosis.

The death receptor CD95 triggers apoptosis upon formation of a death-inducing signaling complex and the activation of caspase-8. Two types of CD95-mediated apoptosis have been distinguished that differ in their efficiency of death-inducing signaling complex formation and the requirement of mitochondria for caspase activation. The validity of the type I/II model, however, has been challenged, as Bcl-2 expression or the use of various CD95 agonists resulted in different apoptosis effects. By identifying a caspase-9-deficient T cell line, we now provide genetic evidence for the two-pathway model of CD95-mediated apoptosis and demonstrate that type II cells strongly depend on caspase-9. Caspase-9-deficient cells revealed strongly impaired apoptosis, caspase activation, and mitochondrial membrane depolarization upon CD95 triggering, whereas, surprisingly, activation of Bak and cytochrome c release were not inhibited. Furthermore, caspase-9-deficient cells did not switch to necrosis, and reconstitution of caspase-9 expression restored CD95 sensitivity. Finally, we also show that different death receptors have a distinct requirement for caspase-9.