Activation of an interleukin 1 converting enzyme-dependent apoptosis pathway by granzyme B.

Cytotoxic T lymphocytes (CTL) can induce apoptosis through a granzyme B-based killing mechanism. Here we show that in cells undergoing apoptosis by granzyme B, both p45 pro-interleukin 1 beta converting enzyme (ICE) and pro-CPP32 are processed. Using ICE deficient (ICE -/-) mice, embryonic fibroblasts exhibit high levels of resistance to apoptosis by granzyme B or granzyme 3, while B lymphoblasts are granzyme B-resistant, thus identifying an ICE-dependent apoptotic pathway that is activated by CTL granzymes. In contrast, an alternative ICE-independent pathway must also be activated as ICE -/- thymocytes remain susceptible to apoptosis by both granzymes. In ICE -/- B cells or HeLa cells transfected with mutant inactive ICE or Ich-1S that exhibit resistance to granzyme B, CPP32 is processed to p17 and poly(ADP-ribose) polymerase is cleaved indicating that this protease although activated was not associated with an apoptotic nuclear phenotype. Using the peptide inhibitor Ac-DEVD-CHO, apoptosis as well as p45 ICE hydrolysis are suppressed in HeLa cells, suggesting that a CPP32-like protease is upstream of ICE. In contrast, p34cdc2 kinase, which is required for granzyme B-induced apoptosis, remains inactive in ICE -/- B cells indicating it is downstream of ICE. We conclude that granzyme B activates an ICE-dependent cell death pathway in some cell types and requires a CPP32-like Ac-DEVD-CHO inhibitable protease acting upstream to initiate apoptosis.

Functional role of interleukin 1 beta (IL-1 beta) in IL-1 beta-converting enzyme-mediated apoptosis.

Prointerleukin-1 beta (pro-IL-1 beta) is the only known physiologic substrate of the interleukin-1 beta (IL-1 beta)-converting enzyme (ICE), the founding member of the ICE/ced-3 cell death gene family. Since secreted mature IL-1 beta has been detected after apoptosis, we investigated whether this cytokine, when produced endogenously, plays a role in cell death. We found that hypoxia-induced apoptosis can be inhibited by either the IL-1 receptor antagonist (IL-1Ra) or by neutralizing antibodies to IL-1 or to its type 1 receptor. IL-1Ra also inhibits apoptosis induced by trophic factor deprivation in primary neurons, as well as by tumor necrosis factor alpha in fibroblasts. In addition, during the G1/S phase arrest, mature IL-1 beta induces apoptosis through a pathway independent of CrmA-sensitive gene activity. We also demonstrate that Ice, when expressed in COS cells, requires the coexpression of pro-IL-1 beta for the induction of apoptosis, which is inhibited by IL-1Ra. Interestingly, we found that mature IL-1 beta has antiapoptotic activity when added exogenously before the onset of hypoxia, which we found is caused in part by its ability to downregulate the IL-1 receptor. Our findings demonstrate that pro-IL-1 beta is a substrate of ICE relevant to cell death, and depending on the temporal cellular commitment to apoptosis, mature IL-1 beta may function as a positive or negative mediator of cell death.

Expression of a specific marker of avian programmed cell death in both apoptosis and necrosis.

Apoptosis and necrosis are two types of cell death with different morphologic features. We report here the isolation of a monoclonal antibody, BV2, that specifically recognizes cells undergoing developmental programmed cell death in different tissues of the chicken and zebra-finch embryos. The antigen recognized by BV2 monoclonal antibody is detected in vitro in primary chicken embryonic fibroblasts induced to die by actinomycin D, as well as fibroblasts induced to die by chemical anoxia. The expression of this specific antigen during necrosis appears to require active protein synthesis. These findings provide evidence that cells from different embryonic tissues undergoing programmed cell death during vertebrate development express similar antigens and indicate that apoptosis and necrosis may share similar biochemical features.

Anti-apogens and anti-engulfens: monoclonal antibodies reveal specific antigens on apoptotic and engulfment cells during chicken embryonic development.

We have isolated a group of monoclonal antibodies that specifically recognize either apoptotic or engulfment cells in the interdigit areas of chicken hind limb foot plates, and throughout the embryo. Ten of these antibodies (anti-apogens) detect epitopes on dying cells that colocalize to areas of programmed cell death, characterized by the presence of apoptotic cells and bodies with typical cellular and nuclear morphology. Our results indicate that cells destined to die, or that are in the process of dying, express specific antigens that are not detectable in or on the surface of living cells. The detection of these apoptotic cell antigens in other areas of programmed cell death throughout the chick embryo indicates that different cell types, which form specific tissues and organs, may utilize similar cell death mechanisms. Six of the monoclonal antibodies (antiengulfens) define a class of engulfment cells which contain various numbers of apoptotic cells and/or apoptotic bodies in areas of programmed cell death. The immunostaining pattern of the anti-engulfen R15F is similar to that of an antibody against a common leukocyte antigen, suggesting the participation of cells from the immune system in the removal of apoptotic cell debris. These novel monoclonal antibody markers for apoptotic and engulfment cells will provide new tools to assist the further understanding of developmental programmed cell death in vertebrates.

Prevention of vertebrate neuronal death by the crmA gene.

Interleukin-1 beta converting enzyme (ICE) is a mammalian homolog of CED-3, a protein required for programmed cell death in the nematode Caenorhabditis elegans. The activity of ICE can be specifically inhibited by the product of crmA, a cytokine response modifier gene encoded by cowpox virus. Microinjection of the crmA gene into chicken dorsal root ganglion neurons was found to prevent cell death induced by deprivation of nerve growth factor. Thus, ICE is likely to participate in neuronal death in vertebrates.

Apoptosis: a different type of cell death.

Apoptosis is a type of cell death that plays an important role in early development and growth of normal adult tissues. It is regulated by physiological stimuli and is present in many species and tissues. The main morphological characteristics are nuclear fragmentation and cellular breakdown in apoptotic vesicles. Internucleosomal DNA fragmentation is an important biochemical feature that is the result of a yet to be isolated endonuclease activity. Experiments using RNA and protein synthesis inhibitors suggest the presence of either intracellular repressors or inducers of apoptosis.

Characterization of uterine epithelium apoptotic cell death kinetics and regulation by progesterone and RU 486.

The authors show here that progesterone suppresses apoptosis, and its antagonist RU 486 induces it in rabbit uterine epithelium, as assessed by morphologic and biochemical studies. The authors' studies demonstrate that internucleosomal DNA fragments are identifiable as early as 24 hours after ovariectomy of pseudopregnant rabbits, and become undetectable 6 days after ovariectomy. Maximal levels of DNA fragmentation (about 74% of total isolated DNA) were observed 36 hours after ovariectomy. The number of apoptotic cells appeared to increase parallel to the increased DNA breakdown, and accounted for approximately 26% of the uterine epithelial cells at 48 hours after ovariectomy. Levels of progesterone in serum dropped precipitously 6 hours after ovariectomy and remained very low for several days. Administration of progesterone, more than any other steroid hormone, to pseudopregnant ovariectomized rabbits, prevented the increase in apoptotic cell death. By contrast, administration of the anti-progestin RU 486 to pseudopregnant rabbits triggered apoptosis, which attained levels similar to those observed in ovariectomized animals. The authors' findings establish that uterine epithelium apoptosis occurs in a time-dependent fashion and provides strong evidence that the actions of progesterone in that tissue are not only to stimulate cell proliferation and differentiation, but also to suppress apoptosis.

Coordinated regulation of apoptosis and cell proliferation by transforming growth factor beta 1 in cultured uterine epithelial cells.

Cell and tissue growth is regulated through a complex interplay of stimulatory and inhibitory signals. We describe two biological actions of transforming growth factor beta 1 (TGF-beta 1) in primary cultures of rabbit uterine epithelial cells: (i) inhibition of cell proliferation and (ii) a concomitant increase in cells undergoing apoptosis (programmed cell death). It is proposed that proliferation and apoptosis together comprise normal cell growth regulation.

Biochemical evidence for programmed cell death in rabbit uterine epithelium.

Uterine epithelial cell proliferation, differentiation, and death are known to be regulated by estrogen and progesterone. The authors investigated a specific pattern of cell death called apoptosis, or programmed cell death, which is biochemically characterized by a specific pattern of DNA degradation. DNA isolated from endometrium of ovariectomized pseudopregnant rabbits showed a pattern of DNA cleavage at internucleosomal locations. In comparison, DNA from the endometrium of non-ovariectomized animals, as well as several other organs, did not exhibit that pattern. This biochemical evidence supports previous and present morphologic data and correlates with it. Under the experimental conditions used, only the uterine epithelial compartment of the endometrium shows apoptotic cell death, which is absent in the stromal compartment.