T cell-derived interferon-γ programs stem cell death in immune-mediated intestinal damage.

Despite the importance of intestinal stem cells (ISCs) for epithelial maintenance, there is limited understanding of how immune-mediated damage affects ISCs and their niche. We found that stem cell compartment injury is a shared feature of both alloreactive and autoreactive intestinal immunopathology, reducing ISCs and impairing their recovery in T cell-mediated injury models. Although imaging revealed few T cells near the stem cell compartment in healthy mice, donor T cells infiltrating the intestinal mucosa after allogeneic bone marrow transplantation (BMT) primarily localized to the crypt region lamina propria. Further modeling with ex vivo epithelial cultures indicated ISC depletion and impaired human as well as murine organoid survival upon coculture with activated T cells, and screening of effector pathways identified interferon-γ (IFNγ) as a principal mediator of ISC compartment damage. IFNγ induced JAK1- and STAT1-dependent toxicity, initiating a proapoptotic gene expression program and stem cell death. BMT with IFNγ-deficient donor T cells, with recipients lacking the IFNγ receptor (IFNγR) specifically in the intestinal epithelium, and with pharmacologic inhibition of JAK signaling all resulted in protection of the stem cell compartment. In addition, epithelial cultures with Paneth cell-deficient organoids, IFNγR-deficient Paneth cells, IFNγR-deficient ISCs, and purified stem cell colonies all indicated direct targeting of the ISCs that was not dependent on injury to the Paneth cell niche. Dysregulated T cell activation and IFNγ production are thus potent mediators of ISC injury, and blockade of JAK/STAT signaling within target tissue stem cells can prevent this T cell-mediated pathology.

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.

A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast.

During apoptosis, proapoptotic factors are released from mitochondria by as yet undefined mechanisms. Patch-clamping of mitochondria and proteoliposomes formed from mitochondrial outer membranes of mammalian (FL5.12) cells has uncovered a novel ion channel whose activity correlates with onset of apoptosis. The pore diameter inferred from the largest conductance state of this channel is approximately 4 nm, sufficient to allow diffusion of cytochrome c and even larger proteins. The activity of the channel is affected by Bcl-2 family proteins in a manner consistent with their pro- or antiapoptotic properties. Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2. Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax. These findings implicate this channel, named mitochondrial apoptosis-induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.

BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis.

Critical issues in apoptosis include the importance of caspases versus organelle dysfunction, dominance of anti- versus proapoptotic BCL-2 members, and whether commitment occurs upstream or downstream of mitochondria. Here, we show cells deficient for the downstream effectors Apaf-1, Caspase-9, or Caspase-3 display only transient protection from "BH3 domain-only" molecules and die a caspase-independent death by mitochondrial dysfunction. Cells with an upstream defect, lacking "multidomain" BAX, BAK demonstrate long-term resistance to all BH3 domain-only members, including BAD, BIM, and NOXA. Comparison of wild-type versus mutant BCL-2, BCL-X(L) indicates these antiapoptotics sequester BH3 domain-only molecules in stable mitochondrial complexes, preventing the activation of BAX, BAK. Thus, in mammals, BH3 domain-only molecules activate multidomain proapoptotic members to trigger a mitochondrial pathway, which both releases cytochrome c to activate caspases and initiates caspase-independent mitochondrial dysfunction.

Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death.

Multiple death signals influence mitochondria during apoptosis, yet the critical initiating event for mitochondrial dysfunction in vivo has been unclear. tBID, the caspase-activated form of a "BH3-domain-only" BCL-2 family member, triggers the homooligomerization of "multidomain" conserved proapoptotic family members BAK or BAX, resulting in the release of cytochrome c from mitochondria. We find that cells lacking both Bax and Bak, but not cells lacking only one of these components, are completely resistant to tBID-induced cytochrome c release and apoptosis. Moreover, doubly deficient cells are resistant to multiple apoptotic stimuli that act through disruption of mitochondrial function: staurosporine, ultraviolet radiation, growth factor deprivation, etoposide, and the endoplasmic reticulum stress stimuli thapsigargin and tunicamycin. Thus, activation of a "multidomain" proapoptotic member, BAX or BAK, appears to be an essential gateway to mitochondrial dysfunction required for cell death in response to diverse stimuli.

Aven, a novel inhibitor of caspase activation, binds Bcl-xL and Apaf-1.

Bcl-x(L), an antiapoptotic Bcl-2 family member, is postulated to function at multiple stages in the cell death pathway. The possibility that Bcl-x(L) inhibits cell death at a late (postmitochondrial) step in the death pathway is supported by this report of a novel apoptosis inhibitor, Aven, which binds to both Bcl-x(L) and the caspase regulator, Apaf-1. Identified in a yeast two-hybrid screen, Aven is broadly expressed and is conserved in other mammalian species. Only those mutants of Bcl-x(L)that retain their antiapoptotic activity are capable of binding Aven. Aven interferes with the ability of Apaf-1 to self-associate, suggesting that Aven impairs Apaf-1-mediated activation of caspases. Consistent with this idea, Aven inhibited the proteolytic activation of caspases in a cell-free extract and suppressed apoptosis induced by Apaf-1 plus caspase-9. Thus, Aven represents a new class of cell death regulator.

Modulation of cell death by Bcl-XL through caspase interaction.

The caspases are cysteine proteases that have been implicated in the execution of programmed cell death in organisms ranging from nematodes to humans. Many members of the Bcl-2 family, including Bcl-XL, are potent inhibitors of programmed cell death and inhibit activation of caspases in cells. Here, we report a direct interaction between caspases and Bcl-XL. The loop domain of Bcl-XL is cleaved by caspases in vitro and in cells induced to undergo apoptotic death after Sindbis virus infection or interleukin 3 withdrawal. Mutation of the caspase cleavage site in Bcl-XL in conjunction with a mutation in the BH1 homology domain impairs the death-inhibitory activity of Bcl-XL, suggesting that interaction of Bcl-XL with caspases may be an important mechanism of inhibiting cell death. However, once Bcl-XL is cleaved, the C-terminal fragment of Bcl-XL potently induces apoptosis. Taken together, these findings indicate that the recognition/cleavage site of Bcl-XL may facilitate protection against cell death by acting at the level of caspase activation and that cleavage of Bcl-XL during the execution phase of cell death converts Bcl-XL from a protective to a lethal protein.

Herpesvirus saimiri encodes a functional homolog of the human bcl-2 oncogene.

Here we demonstrate that open reading frame 16 (ORF16) of the oncogenic herpesvirus saimiri protects cells from heterologous virus-induced apoptosis. The BH1 and BH2 homology domains are highly conserved in ORF16, and ORF16 heterodimerizes with Bcl-2 family members Bax and Bak. However, ORF16 lacks the core sequence of the conserved BH3 homology domain, suggesting that this region is not essential for anti-apoptotic activity. Conservation of a functional bcl-2 homolog among gammaherpesviruses suggests that inhibition of programmed cell death is important in the biology of these viruses.

A Bcl-2 homolog encoded by Kaposi sarcoma-associated virus, human herpesvirus 8, inhibits apoptosis but does not heterodimerize with Bax or Bak.

The Bcl-2 protein family is characterized by the ability to modulate cell death, and members of this family share two highly conserved domains called Bcl-2 homology 1 (BH1) and 2 (BH2) which have been shown to be critical for the death-repressor activity of Bcl-2 and Bcl-xL. Through sequence analysis we identified a novel viral Bcl-2 homolog, designated KSbcl-2, from human herpesvirus 8 (HHV8) or Kaposi sarcoma-associated herpesvirus. The overall amino acid sequence identity between KSbcl-2 and other Bcl-2 homologs is low (15-20%) but concentrated within the BH1 and BH2 regions. Overexpression of KSbcl-2 blocked apoptosis as efficiently as Bcl-2, Bcl-xL, or another viral Bcl-2 homolog encoded by Epstein-Barr virus, BHRF1. Interestingly, KS-bcl-2 neither homodimerizes nor heterodimerizes with other Bcl-2 family members, suggesting that KSbcl-2 may have evolved to escape any negative regulatory effects of the cellular Bax and Bak proteins. Furthermore, the herpesvirus Bcl-2 homologs including KSbcl-2, BHRF1, and ORF16 of herpesvirus saimiri contain poorly conserved Bcl-2 homology 3 (BH3) domains compared with other mammalian Bcl-2 homologs, implying that BH3 may not be essential for anti-apoptotic function. This is consistent with our observation that amino acid substitutions within the BH3 domain of Bcl-xL had no effect on its death-suppressor activity.

Conversion of Bcl-2 to a Bax-like death effector by caspases.

Caspases are a family of cysteine proteases implicated in the biochemical and morphological changes that occur during apoptosis (programmed cell death). The loop domain of Bcl-2 is cleaved at Asp34 by caspase-3 (CPP32) in vitro, in cells overexpressing caspase-3, and after induction of apoptosis by Fas ligation and interleukin-3 withdrawal. The carboxyl-terminal Bcl-2 cleavage product triggered cell death and accelerated Sindbis virus-induced apoptosis, which was dependent on the BH3 homology and transmembrane domains of Bcl-2. Inhibitor studies indicated that cleavage of Bcl-2 may further activate downstream caspases and contribute to amplification of the caspase cascade. Cleavage-resistant mutants of Bcl-2 had increased protection from interleukin-3 withdrawal and Sindbis virus-induced apoptosis. Thus, cleavage of Bcl-2 by caspases may ensure the inevitability of cell death.

Bax-independent inhibition of apoptosis by Bcl-XL.

The Bcl-2-related protein, Bcl-XL, has been shown to block apoptosis induced by a variety of stimuli and to be a stronger protector against apoptosis than Bcl-2 under certain circumstances. Using site-specific mutagenesis, we show here that the amino-acid residues critical for protection of cells by Bcl-XL against Sindbis virus-induced apoptosis are clustered within the Bcl-2-homology regions 1 and 2 (BH1 and BH2 regions). The residues necessary for Bcl-XL function are not identical to those required for Bcl-2 function. Although it has been suggested that heterodimerization between Bcl-XL and Bax is essential for the anti-death activity of Bcl-XL (refs 7,8), our results suggest that the interaction with Bax is not required for Bcl-XL to exert its death-repressing activity. Specific mutations that disrupt the ability of Bcl-XL to interact with Bax or Bak still preserve 70-80% of the anti-death activity of wild-type Bcl-XL.

Calmodulin-dependent protein kinases in rat glioblastoma.

The mitogenic activity of several growth factors is mediated by calcium-dependent signal transduction. Calmodulin (CaM) binding proteins such as CaM-dependent protein kinases are important components of this pathway and may be altered in diseases characterized by abnormal cell growth. CaM kinase II is believed to regulate the phosphorylation of microtubular-associated proteins and control the initiation of DNA synthesis. Furthermore, drugs that inhibit CaM-mediated signal transduction also inhibit cellular proliferation and are cytotoxic to numerous malignant cell lines, including those established from malignant gliomas. Yet, little is known about CaM-dependent protein kinases in these tumors. Therefore, we have investigated the activity and distribution of CaM-dependent protein kinase II in normal and malignant glial tissues, a kinase believed to play a critical role in cell cycle regulation. C6 and 9L cells contained kinase activities that were activated by Ca2+/CaM and inhibited by trifluoperazine. Tissue extracts from these cell lines and from rat brain white matter phosphorylated exogenous synapsin I in a pattern consistent with the presence of CaM kinase II activity as determined by phosphopeptide mapping. CaM kinase II activity was confirmed using a specific peptide substrate and inhibitor. An unexpected finding was that glioma lines, but not rat brain white matter, also contained a CaM-dependent protein kinase detected by the phosphorylation of a M(r) 100,000 protein, subsequently identified as elongation factor 2, the only known substrate for CaM kinase III.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation of elongation factor 2 in normal and malignant rat glial cells.

Certain calmodulin (CaM)-dependent protein kinases phosphorylate substrates have been implicated in regulating cellular proliferation. In this study, CaM-dependent phosphorylation has been examined in normal and tumor tissue from rat brain to determine whether differences exist. Using in vitro phosphorylation reactions, we compared endogenous substrates for Ca2+/CaM-dependent protein kinases in rat brain white matter (RBWM), a tissue rich in normal glia, to those of C6 rat glioma cells. A major phosphoprotein having a M(r) of 100,000 was observed in proliferating C6 cells that was not present in RBWM or in nonproliferating cells. Phosphorylation was stimulated by Ca2+ and CaM and inhibited by trifluoperazine. An antibody to elongation factor 2 (EF-2) immunoprecipitated the M(r) 100,000 protein from C6 cells. EF-2 was present in RBWM but was not phosphorylated. Homogenates of RBWM did not phosphorylate exogenous EF-2, which suggested the absence of CaM kinase III activity in normal glial tissue. Furthermore, the addition of purified, exogenous CaM kinase III to homogenates of RBWM resulted in EF-2 phosphorylation. These data demonstrate that a basal level of EF-2 phosphorylation exists in proliferating glioma cells that is markedly diminished or absent in normal glial tissue and is due to the activity of CaM kinase III.

Persistence of intestinal antibody response to heterologous rotavirus infection in a murine model beyond 1 year.

We used an ELISPOT (enzyme-linked immunosorbent spot) assay to quantitate the long-term rotavirus-specific intestinal antibody response in a murine model. The frequency of murine intestinal antibody-secreting cells (ASCs) was followed for a period of 1 year after a single dose of rhesus rotavirus (10(6) PFU) was administered at 10 days of age. Some animals were boosted at that time with a second dose. One year after infection, virus-specific ASCs declined from acute-phase levels, but they were still present at significant levels (1.32 x 10(4) virus-specific ASCs per 10(6) intestinal mononuclear cells; approximately 17% of the previously reported response at 1 month after infection). A booster dose 1 year after the primary infection produced a 100% increase in virus-specific ASCs but did not restore the response to that of the primary infection.

"Postgastrectomy" bezoar secondary to gastric cancer.

Phytobezoars are frequently associated with partial gastrectomy and are generally considered benign. However, we report two patients who developed a gastric bezoar due to malignancy at the anastomotic site. Development of intragastric bezoar may be an indication of neoplastic growth sufficient to obstruct a small gastric outlet. Consequently, we conclude that endoscopic or surgical evaluation of the anastomotic site is necessary in patients who develop bezoars after gastric surgery.

Recombinant baculovirus-expressed rotavirus protein (VP4) in an ELISPOT assay of antibody secretion.

Studies on the protein specificity of the intestinal antibody response to rotavirus infection have been hampered by lack of antigenically conserved isolated proteins to serve as antigens in immunochemical assays. In this report, the use of an antigenically conserved baculovirus-expressed rotavirus protein (VP4) as a capture antigen in the ELISPOT assay is described. Anti-VP4 antibody-secreting hybridoma cells are used as a test population to show that expressed VP4 as the capture antigen detects numbers of antibody secreting cells comparable to intact rotavirus particles. Hybridoma cells specific for other rotavirus proteins are used to ensure the specificity of the expressed VP4 in the assay. The flexibility and ease of use of a recombinant expressed protein product as a capture antigen in this assay dramatically enhances the ability to quantitate intestinal antibody responses to specific viral proteins.

Murine intestinal antibody response to heterologous rotavirus infection.

Rotavirus is the most important worldwide cause of severe gastroenteritis. Extensive efforts have been devoted to the design of a vaccine that will prevent disease, but development of a more effective vaccine strategy may require progress in the understanding of the mucosal immune response to replicating viral antigens. In this article, we report the characterization of the intestinal antibody response of a murine model to heterologous infection with the rhesus rotavirus vaccine strain. We have adapted the enzyme-linked immunospot assay to measure this response without the difficulties associated with measurement of antibodies in intestinal contents or the artifacts associated with culturing of lymphocytes. The predominant response in terms of antibody-secreting cells (ASC) is seen in the small intestine lamina propria, which can be measured within 4 days of infection, peaks 3 weeks after infection, and remains near that level for longer than 8 weeks. The magnitude of the immunoglobulin A (IgA) cell response is approximately 10 times greater than the intestinal IgG cell response, and IgM cells are rare. Virus-specific ASC constitute approximately 50% of all ASC in the gut at the peak of the virus-specific response. This response is considerably greater than responses to nonreplicating mucosal antigens measured by similar techniques. Enteral infection engenders minimal virus-specific ASC response in the spleen. Rhesus rotavirus-specific enzyme-linked immunosorbent assay and neutralization assays of serum and intestinal contents did not correlate with virus-specific ASC response.

Effects of bradykinin on intracellular calcium regulation in human ciliated airway epithelium.

The Ca(2+)-mobilizing action of bradykinin (BK) was investigated in ciliated human nasal epithelial (HNE) cells utilizing fura-2 fluorescence and microspectrofluorimetry. In ciliated cells, basal intracellular Ca2+ concentration ([Ca2+]i) was 123 +/- 3 nM (n = 142). BK caused [Ca2+]i to increase (spike) rapidly (within 6 s) to greater than 550 nM by releasing Ca2+ from intracellular pools. The mean effective dose for the process was 1.5 x 10(-7) M BK. The spike was due to the activation of a beta 2-receptor. The spike was unaffected by inhibitors of either cyclooxygenase or voltagegated Ca2+ channels. After the spike, [Ca2+]i decreased to a plateau level (120-250 nM). This plateau persisted (up to 10 min) until the addition of La3+ (0.3 x 10(-3) M) or until the removal of either extracellular Ca2+ or the agonist. No changes in adenosine 3',5'-cyclic monophosphate (cAMP) levels were detected after BK exposure. Additional studies revealed that indomethacin (10(-6) M), isoproterenol (10(-5) M), forskolin (10(-5) M), and dibutyryl cAMP (1 mM) had no effect on [Ca2+]i in ciliated HNE cells. In summary, these data suggest that 1) BK mediates both Ca2+ release from internal pools and Ca2+ entry into the cytoplasm from the extracellular space, and 2) unlike the response to cultured dog airway epithelia, the release of [Ca2+]i in response to BK does not appear to be mediated by either cyclooxygenase pathway or adenylate cyclase-cAMP systems.