Christopher J. Froelich

Pro-caspase-3 Is a Major Physiologic Target of Caspase-8

The apoptotic signal triggered by ligation of members of the death receptor family is promoted by sequential activation of caspase zymogens. We show here that in a purified system, the initiator caspases-8 and -10 directly process the executioner pro-caspase-3 with activation rates (k cat/K m ) of 8.7 × 105 and 2.8 × 105 m −1 s−1, respectively. These rates are of sufficient magnitude to indicate direct processingin vivo. Differentially processed forms of caspase-3 that accumulate during its activation have similar rates of activation, activities, and specificities. The pattern and rate of caspase-8 induced activation of pro-caspase-3 in cytosolic extracts was the same as in a purified system. Moreover, immunodepletion of a putative intermediary in the pathway to activation, pro-caspase-9, was without consequence. Taken together these data demonstrate that the initiator caspase-8 can directly activate pro-caspase-3 without the requirement for an accelerator. The in vitro data thus help to deconvolute previous in vivo transfection studies which have debated the role of a direct versus indirect transmission of the apoptotic signal generated by ligation of death receptors.

The CED-3/ICE-like Protease Mch2 Is Activated during Apoptosis and Cleaves the Death Substrate Lamin A

Phylogenetic analysis of the CED-3/ICE family of cysteine proteases suggests the existence of a subfamily most related to the Caenorhabditis elegans death gene ced-3 and includes Yama (CPP32, apopain), LAP3 (Mch3, CMH1), and Mch2. Here, we show that Mch2 is processed from its zymogen form to a proteolytically active dimeric species during execution of the apoptotic program and by the cytotoxic T cell death protease granzyme B. Additionally, like Yama and LAP3, Mch2 functions downstream of the death inhibitors Bcl-2, Bcl-xL, and CrmA. Importantly, Mch2, but not Yama or LAP3, is capable of cleaving lamin A to its signature apoptotic fragment, indicating that Mch2 is an apoptotic laminase.

New paradigm for lymphocyte granule-mediated cytotoxicity: Target cells bind and internalize granzyme B, but an endosomolytic agent is necessary for cytosolic delivery and subsequent apoptosis

Lymphocyte granule-mediated apoptosis is postulated to entail the formation of membrane pores by perforin. Then soluble granzyme reaches the cytosol either through these pores or by reparative pinocytosis. We demonstrate here that Jurkat cells bind and internalize granzyme B via high affinity binding sites without toxic consequence. Apoptosis occurs, however, if sublytic perforin is added to targets washed free of soluble granzyme B. We suggest that granule-mediated apoptosis mimics viral strategies for cellular entry. Accordingly, co-internalization of granzyme B with adenovirus, a virus that escapes endosomes to reach the cytosol, also induced apoptosis. Poly(ADP-ribose) polymerase cleavage and processing of CPP32, ICE-LAP3, and Mch2 were detected at 30 min, while cytosolic acidification and DNA fragmentation occurred at 60 min. Annexin V binding and membrane permeabilization arose at 4 h. The concurrent activation of the Ced-3 proteases differed from the rate at which each cysteine protease is cleaved in vitro by granzyme B. Thus, granzyme B may not directly process these proteases in whole cells but rather may function by activating a more proximal enzyme. These results indicate that adenovirus-mediated delivery of granzyme B is suitable for elucidating biochemical events that accompany granule-mediated apoptosis.

ICE-LAP6, a novel member of the ICE/Ced-3 gene family, is activated by the cytotoxic T cell protease granzyme B.

Members of the ICE/Ced-3 gene family are likely effector components of the cell death machinery. Here, we characterize a novel member of this family designated ICE-LAP6. By phylogenetic analysis, ICE-LAP6 is classified into the Ced-3 subfamily which includes Ced-3, Yama/CPP32/apopain, Mch2, and ICE-LAP3/Mch3/CMH-1. Interestingly, ICE-LAP6 contains an active site QACG pentapeptide, rather than the QACG pentapeptide shared by other family members. Overexpression of ICE-LAP6 induces apoptosis in MCF7 breast carcinoma cells. More importantly, ICE-LAP6 is proteolytically processed into an active cysteine protease by granzyme B, an important component of cytotoxic T cell-mediated apoptosis. Once activated, ICE-LAP6 is able to cleave the death substrate poly(ADP-ribose) polymerase into signature apoptotic fragments.

Cytotoxic cell granule-mediated apoptosis: Perforin delivers granzyme b-serglycin complexes into target cells without plasma membrane pore formation

The mechanism underlying perforin (PFN)-dependent delivery of apoptotic granzymes during cytotoxic cell granule-mediated death remains speculative. Granzyme B (GrB) and perforin were found to coexist as multimeric complexes with the proteoglycan serglycin (SG) in cytotoxic granules, and cytotoxic cells were observed to secrete exclusively macromolecular GrB-SG. Contrary to the view that PFN acts as a gateway for granzymes through the plasma membrane, monomeric PFN and, strikingly, PFN-SG complexes were shown to mediate cytosolic delivery of macromolecular GrB-SG without producing detectable plasma membrane pores. These results indicate that granule-mediated apoptosis represents a phenomenon whereby the target cell perceives granule contents as a multimeric complex consisting of SG, PFN, and granzymes, which are, respectively, the scaffold, translocator, and targeting/informational components of this modular delivery system.

Cytosolic Delivery of Granzyme B by Bacterial Toxins: Evidence that Endosomal Disruption, in Addition to Transmembrane Pore Formation, Is an Important Function of Perforin

ABSTRACT Granule-mediated cell killing by cytotoxic lymphocytes requires the combined actions of a membranolytic protein, perforin, and granule-associated granzymes, but the mechanism by which they jointly kill cells is poorly understood. We have tested a series of membrane-disruptive agents including bacterial pore-forming toxins and hemolytic complement for their ability to replace perforin in facilitating granzyme B-mediated cell death. As with perforin, low concentrations of streptolysin O and pneumolysin (causing <10%51Cr release) permitted granzyme B-dependent apoptosis of Jurkat and Yac-1 cells, but staphylococcal alpha-toxin and complement were ineffective, regardless of concentration. The ensuing nuclear apoptotic damage was caspase dependent and included cleavage of poly(ADP-ribose) polymerase, suggesting a mode of action similar to that of perforin. The plasma membrane lesions formed at low dose by perforin, pneumolysin, and streptolysin did not permit diffusion of fluorescein-labeled proteins as small as 8 kDa into the cell, indicating that large membrane defects are not necessary for granzymes (32 to 65 kDa) to enter the cytosol and induce apoptosis. The endosomolytic toxin, listeriolysin O, also effected granzyme B-mediated cell death at concentrations which produced no appreciable cell membrane damage. Cells pretreated with inhibitors of endosomal trafficking such as brefeldin A took up granzyme B normally but demonstrated seriously impaired nuclear targeting of granzyme B when perforin was also added, indicating that an important role of perforin is to disrupt vesicular protein trafficking. Surprisingly, cells exposed to granzyme B with perforin concentrations that produced nearly maximal 51Cr release (1,600 U/ml) also underwent apoptosis despite excluding a 8-kDa fluorescein-labeled protein marker. Only at concentrations of >4,000 U/ml were perforin pores demonstrably large enough to account for transmembrane diffusion of granzyme B. We conclude that pore formation may allow granzyme B direct cytosolic access only when perforin is delivered at very high concentrations, while perforin’s ability to disrupt endosomal trafficking may be crucial when it is present at lower concentrations or in killing cells that efficiently repair perforin pores.

Human and Mouse Granzyme A Induce a Proinflammatory Cytokine Response

Granzyme A (GzmA) is considered a major proapoptotic protease. We have discovered that GzmA-induced cell death involves rapid membrane damage that depends on the synergy between micromolar concentrations of GzmA and sublytic perforin (PFN). Ironically, GzmA and GzmB, independent of their catalytic activity, both mediated this swift necrosis. Even without PFN, lower concentrations of human GzmA stimulated monocytic cells to secrete proinflammatory cytokines (interleukin-1beta [IL-1beta], TNFalpha, and IL-6) that were blocked by a caspase-1 inhibitor. Moreover, murine GzmA and GzmA(+) cytotoxic T lymphocytes (CTLs) induce IL-1beta from primary mouse macrophages, and GzmA(-/-) mice resist lipopolysaccharide-induced toxicity. Thus, the granule secretory pathway plays an unexpected role in inflammation, with GzmA acting as an endogenous modulator.

Extracellular matrix remodeling by human granzyme B via cleavage of vitronectin, fibronectin, and laminin

Human granzyme B (GrB) released from cytotoxic lymphocytes plays a key role in the induction of target cell apoptosis when internalized in the presence of perforin. Here we demonstrate that GrB also possesses a potent extracellular matrix remodeling activity. Both native and recombinant GrB caused detachment of immortalized and transformed cell lines, primary endothelial cells, and chondrocytes. Cell detachment by GrB induced endothelial cell death (anoikis). GrB also inhibited tumor cell spreading, migration, and invasion in vitro. Investigation into the underlying mechanism revealed that GrB efficiently cleaves three proteins involved in extracellular matrix structure and function: vitronectin, fibronectin, and laminin. In vitronectin, GrB cleaves after an Arg-Lys-Asp (RGD) motif, which is part of the integrin-binding site found in matrix proteins. We propose that targeting of the integrin-extracellular matrix interface by GrB may allow perforin-independent killing of target cells via anoikis, restrict motility of tumor cells, facilitate lymphocyte migration, or directly reduce virus infectivity. It may also contribute to tissue destruction in diseases in which extracellular GrB is evident, such as rheumatoid arthritis and atherosclerosis.

Sustained Delivery of Interleukin-2 from a Poloxamer 407 Gel Matrix Following Intraperitoneal Injection in Mice

Parenteral delivery of recombinant biologic response modifiers (BRMs) remains a challenge because of the brief intravascular half-life of most recombinant proteins and their associated rapid clearance from the circulation. Recombinant derived interleukin-2 (rIL-2) was formulated with Pluronic F-127, N.F. (poloxamer 407, N.F.) and the biological activity determined vs time at 4, 22, and 37°C. As assessed by rIL-2-induced peripheral blood lymphocyte (PBL) uptake of [3H]thymidine, storage of rIL-2/poloxamer 407 (33% w/w) for 72 hr at 4 and 22°C did not result in an overall negative slope of the [3H]thymidine vs time profiles. However, storage of an rlL-2/poloxamer formulation at 37°C for 72 hr resulted in an approximate 15% reduction in the biological activity as assessed by [3H]thymidine incorporation. As assessed by bioassay ([3H]thymidine uptake), the cumulative percentage rIL-2 released in vitro at 22°C after 8 hr from rIL-2/poloxamer 407 matrices containing either 30% (w/w) or 35% (w/w) poloxamer 407 was 81.8 ± 1.7 and 82.1 ± 4.7%, respectively. When ELISA was used to determine the amount of rIL-2 released vs time, the corresponding values for the cumulative percentage rIL-2 released were 82.6 ± 10.1 and 40.9 ± 8.8%. Cytotoxicity of rIL-2-stimulated PBLs cultured with poloxamer 407 (0.17%, w/w) toward malignant Daudi cells was significantly (P < 0.05) enhanced compared to controls. Finally, mice injected with the rIL-2/poloxamer 407 formulation (1 × 105 U/inj. q.d. × 3 days) demonstrated a bioequivalent effect of rIL-2-induced natural killer (NK) cell activity in vitro toward malignant murine YAC-1 cells at one-half the standard exogenously administered dose of rIL-2 known to generate enhanced NK lytic activity in mice (1 × 105 U/inj. b.i.d. x 3 days). No untoward systemic side effects were observed for mice injected i.p. with polymer vehicle alone (30%, w/w) (0.15 ml q.d. × 3 days), pH 7 phosphate-buffered saline (PBS) (0.15 ml q.d. × 3 days), rIL-2 formulated with poloxamer 407 (30%, w/w) (1 × 105 U/0.15 ml q.d. × 3 days and 0.5 × 105 U/0.15 ml q.d. × 3 days), or rIL-2 dissolved in PBS (1 × 105 U/0.15 ml b.i.d. × 3 days). Thus, poloxamer 407, N.F., did not denature rIL-2 when the latter was stabilized with human serum albumin (HSA), enhanced the in vitro lytic ability of human rIL-2-stimulated PBLs against malignant Daudi cells, and served as a sustained-release parenteral vehicle for rIL-2 when injected i.p. into mice. Thus, based on these preliminary findings, it appears that poloxamer 407, N.F., may potentially be useful for the formulation and sustained delivery of select protein pharmaceuticals following extravascular administration.

Lymphocyte granule-mediated apoptosis: matters of viral mimicry and deadly proteases

Abstract A new form of intercellular signaling is described for lymphocyte granule-mediated apoptosis. Christopher Froelich, Vishva Dixit and Xiaohe Yang propose that perforin delivers granzyme B into target cells by a mechanism analogous to receptor-dependent endocytosis of pathogens. Once in the cytosol, granzyme B prefers to initiate apoptosis by activating the apical protease caspase 10.