Caspase 8 activity in membrane blebs after anti-Fas ligation.

Previous studies of thymocyte apoptosis using a series of cell-permeable fluorogenic peptide substrates showed that Fas cross-linking triggered a caspase cascade in which cleavage of the IETDase (caspase 8-selective) substrate was the earliest caspase activity measured by flow cytometry. This result was expected in light of the abundant evidence for caspase 8 activation as an initiating event in the Fas death pathway. However, when apoptosis was induced by anti-Fas in CTL and the caspase cascade examined by this approach, IETDase activation followed increases in LEHDase, YVHDase, and VEIDase activities (selective for caspases 9, 1, and 6, respectively). When examined by confocal microscopy, anti-Fas-treated CTL showed the early appearance of IETDase-containing plasma membrane vesicles and their release from the CTL surface, followed by activation of other caspase activities in the cell interior. Since these vesicles were not included in the flow cytometry analysis, the early IETDase activity had been underestimated. In contrast to anti-Fas, induction of apoptosis in these CTL by IL-2 withdrawal resulted in early IETDase activity in the cytoplasm, with no plasma membrane vesiculation. Thus, anti-Fas-induced initiation of caspase activity at the plasma membrane may in some cells result in local proteolysis of submembrane proteins, leading to generation of membrane vesicles that are highly enriched in active caspase 8.

Defective granule exocytosis in Rab27a-deficient lymphocytes from Ashen mice.

Because mutations in Rab27a have been linked to immune defects in humans, we have examined cytotoxic lymphocyte function in ashen mice, which contain a splicing mutation in Rab27a. Ashen cytotoxic T lymphocytes (CTLs) showed a >90% reduction in lytic activity on Fas-negative target cells compared with control C3H CTLs, and ashen natural killer cell activity was likewise diminished. Although their granule-mediated cytotoxicity pathway is profoundly defective, ashen CTLs displayed a normal FasL-Fas cytotoxicity pathway. The CD4/8 phenotype of ashen T cells and their proliferative responses were similar to controls. Ashen CTLs had normal levels of perforin and granzymes A and B and normal-appearing perforin-positive granules, which polarized upon interaction of the CTLs with anti-CD3-coated beads. However, rapid anti-CD3-induced granule secretion was drastically defective in both CD8(+) and CD4(+) T cells from ashen mice. This defect in exocytosis was not observed in the constitutive pathway, as T cell receptor-stimulated interferon-gamma secretion was normal. Based on these results and our demonstration that Rab27a colocalizes with granzyme B-positive granules and is undetectable in ashen CTLs, we conclude that Rab27a is required for a late step in granule exocytosis, compatible with current models of Rab protein function in vesicle docking and fusion.

Beta-chemokines inhibit activation-induced death of lymphocytes from HIV-infected individuals.

The present study investigates the role of the HIV-suppressive beta-chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1 and RANTES in activation-induced cell death (AICD). A pool of these beta-chemokines reduced anti-CD3-induced apoptosis of T cell blasts from healthy blood donors in a dose-dependent manner. Although the pooled beta-chemokines were more effective, the inhibitory effect could also be mediated by each of the individual chemokines and was blocked by neutralizing anti-chemokine antibodies. The beta-chemokines also inhibited pokeweed mitogen/staphylococcal enterotoxin B-induced T lymphocyte apoptosis in 33/49 HIV-infected (HIV+) individuals. This anti-apoptotic effect was not correlated with the patients' CD4 T cell counts. beta-chemokines did not lead to altered secretion of IL-2, IL-4, IFN-gamma or IL-10 in response to activation stimuli in either normal T cell blasts or peripheral blood mononuclear cells from HIV+ individuals. Co-incubation with beta-chemokines did not inhibit anti-CD3-induced expression of cell surface Fas ligand, nor did it alter levels of the death receptor Fas or Bcl-2 in T cell blasts, suggesting that the beta-chemokines are blocking AICD downstream of Fas. These observations indicate that beta-chemokines may play a novel role as modulators of AICD, in addition to their known role as chemoattractants and inhibitors of HIV replication.

Assessment of caspase activities in intact apoptotic thymocytes using cell-permeable fluorogenic caspase substrates.

To detect caspase activities in intact apoptotic cells at the single cell level, cell-permeable fluorogenic caspase substrates were synthesized incorporating the optimal peptide recognition motifs for caspases 1, 3/7, 6, 8, and 9. Caspase activities were then assessed at various times after in vitro treatment of mouse thymocytes with dexamethasone or anti-Fas antibody. Dexamethasone induced the following order of appearance of caspase activities as judged by flow cytometry: LEHDase, WEHDase, VEIDase, IETDase, and DEVDase. Since the relative order of caspases 3 (DEVDase) and 6 (VEIDase) in the cascade has been controversial, this caspase activation order was reexamined using confocal microscopy. The VEIDase activity appeared before DEVDase in every apoptotic cell treated with dexamethasone. In contrast, anti-Fas stimulation altered this sequence: IETDase was the first measurable caspase activity and DEVDase preceded VEIDase. In an attempt to determine the intracellular target of the potent antiapoptotic agent carbobenzoxy-valyl-alanyl-aspartyl(beta-methyl ester)-fluoromethyl ketone (Z-VAD[OMe]-FMK), we examined its ability to inhibit previously activated intracellular caspases. However, no significant reductions of these activities were observed. These fluorogenic caspase substrates allow direct observation of the caspase cascade in intact apoptotic cells, showing that the order of downstream caspase activation is dependent on the apoptotic stimulus.

Posttranslational regulation of the retinoblastoma gene family member p107 by calpain protease.

The retinoblastoma protein plays a critical role in regulating the G1/S transition. Less is known about the function and regulation of the homologous pocket protein p107. Here we present evidence for the posttranslational regulation of p107 by the Ca2+-activated protease calpain. Three negative growth regulators, the HMG-CoA reductase inhibitor lovastatin, the antimetabolite 5-fluorouracil, and the cyclic nucleotide dibutyryl cAMP were found to induce cell type-specific loss of p107 protein which was reversible by the calpain inhibitor leucyl-leucyl-norleucinal but not by the serine protease inhibitor phenylmethylsulfonylfluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26S proteasome. Purified calpain induced Ca2+-dependent p107 degradation in cell lysates. Transient expression of the specific calpain inhibitor calpastatin blocked the loss of p107 protein in lovastatin-treated cells, and the half-life of p107 was markedly lengthened in lovastatian-treated cells stably transfected with a calpastatin expression vector versus cells transfected with vector alone. The data presented here demonstrate down-regulation of p107 protein in response to various antiproliferative signals, and implicate calpain in p107 posttranslational regulation.

Nitric oxide synthase plays a signaling role in TCR-triggered apoptotic death.

A functional role for stimulated nitric oxide (NO) production was tested in the TCR-triggered death of mature T lymphocytes. In purified peripheral human T cell blasts or the 2B4 murine T cell hybridoma, apoptotic cell death induced by immobilized anti-CD3 was blocked by inhibitors of NO synthase (NOS) in a stereospecific and concentration-dependent manner. This effect appeared to be selective since apoptotic death induced by anti-Fas Ab or the steroid dexamethasone was not affected by NOS inhibitors. TCR-stimulated expression of functional Fas ligand was attenuated in a stereospecific manner by NOS inhibitors, but these compounds did not inhibit TCR-stimulated IL-2 secretion or CD69 surface expression. Nitrosylated tyrosines, a stable marker for NO generation, were immunochemically detected in T cells using flow cytometry. TCR signals induced NO production, as measured by an increase in nitrotyrosine-specific staining. NOS enzymatic activity was detected in lysates of 2B4 cells, and Western blot analysis suggests that the activity is due to expression of the neuronal isoform of NOS. Thus, T cells have the capacity to generate NO upon Ag signaling, which may affect signal transduction, Fas ligand surface expression, and apoptotic cell death of mature T lymphocytes.

Inducible nonlymphoid expression of Fas ligand is responsible for superantigen-induced peripheral deletion of T cells.

Fas (CD95) and Fas ligand (FasL) play major roles in staphylococcal enterotoxin B (SEB)-induced peripheral deletion of Vbeta8+ T cells. We found that peripheral deletion was defective in radiation chimeras with non-functional tissue FasL, regardless of the FasL status of the bone marrow-derived cells. SEB induced a dramatic upregulation of FasL expression and function in nonlymphoid cells of liver and small intestine. This effect was resistant to inhibition by cyclosporin A, which also failed to inhibit peripheral deletion. In SCID animals nonlymphoid tissues did not express FasL in response to SEB unless transplanted lymphocytes were present. Thus, some immune responses induce FasL in nonlymphoid tissues, which in turn kills activated lymphocytes, leading to peripheral T cell deletion.

Supraoptimal peptide-major histocompatibility complex causes a decrease in bc1-2 levels and allows tumor necrosis factor alpha receptor II-mediated apoptosis of cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTLs) are primary mediators of viral clearance, but high viral burden can result in deletion of antigen-specific CTLs. We previously reported a potential mechanism for this deletion: tumor necrosis factor (TNF)-alpha-mediated apoptosis resulting from stimulation with supraoptimal peptide-major histocompatibility complex. Here, we show that although death is mediated by TNF-alpha and its receptor (TNF-RII), surprisingly neither the antigen dose dependence of TNF-alpha production nor that of TNF-RII expression can account for the dose dependence of apoptosis. Rather, a previously unrecognized effect of supraoptimal antigen in markedly decreasing levels of the antiapoptotic protein Bc1-2 was discovered and is likely to account for the gain in susceptibility or competence to sustain the death signal through TNF-RII. This decrease requires a signal through the TCR, not just through TNF-RII. Although death mediated by TNF-RII is not as widely studied as that mediated by TNF-RI, we show here that it is also dependent on proteolytic cleavage by caspases and triggered by a brief initial encounter with antigen. These results suggest that determinant density can regulate the immune response by altering the sensitivity of CTLs to the apoptotic effects of TNF-alpha by decreasing Bc1-2 levels.

Caspase dependence of target cell damage induced by cytotoxic lymphocytes.

Since the CTL secreted granule protease granzyme B can activate multiple target caspases, it has been proposed that this pathway is responsible for CTL-induced cytolysis of Fas-negative targets. However, target lysis via the granule exocytosis pathway is completely resistant to caspase inhibitors. To test the possibility that granzymes trigger a postcaspase cytoplasmic apoptotic pathway leading to lysis, we have examined the caspase dependence of several cytoplasmic changes associated with apoptotic death. Rapid prelytic phosphatidylserine externalization was induced in Jurkat target cells by both the Fas ligand (FasL)/Fas and the granule exocytosis effector pathways. This was specifically blocked by peptide ketone caspase inhibitors when induced by the former, but not by the latter, pathway. A rapid prelytic loss of target mitochondrial psi was also induced by both CTL effector pathways, and this was also specifically blocked by caspase inhibitors when induced by the FasL/Fas, but not by the granule exocytosis, pathway. Similarly, target membrane blebbing induced by CTL via the FasL/Fas, but not via the granule exocytosis, effector pathway was specifically blocked by caspase inhibitors. In contrast to the above nonnuclear damage, CTL-induced target staining by the lipid probe FM1-43 reflecting plasma membrane endocytosis was blocked by caspase inhibitors. Thus, when caspase activation is blocked, the granule exocytosis pathway triggers several parameters of target apoptotic damage in addition to lysis, suggesting that granzymes directly trigger a postcaspase cytoplasmic apoptotic death pathway.

Regulation of cyclin D1 by calpain protease.

Cyclin D1, a critical positive regulator of G1 progression, has been implicated in the pathogenesis of certain cancers. Regulation of cyclin D1 occurs at the transcriptional and posttranscriptional level. Here we present evidence that cyclin D1 levels are regulated at the posttranscriptional level by the Ca2+-activated protease calpain. Serum starvation of NIH 3T3 cells resulted in rapid loss of cyclin D1 protein that was completely reversible by calpain inhibitors. Actinomycin D and lovastatin induced rapid loss of cyclin D1 in prostate and breast cancer cells that was reversible by calpain inhibitors and not by phenylmethylsulfonyl fluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26 S proteasome. Treatment of intact NIH 3T3, prostate, and breast cancer cells with a calpain inhibitor dramatically increased the half-life of cyclin D1 protein. Addition of purified calpain to PC-3-M lysates resulted in Ca2+-dependent cyclin D1 degradation. Transient expression of the calpain inhibitor calpastatin increased cyclin D1 protein in serum-starved NIH 3T3 cells. Cyclins A, E, and B1 have been reported to be regulated by proteasome-associated proteolysis. The data presented here implicate calpain in cyclin D1 posttranslational regulation.

Functional cleavage of the common cytokine receptor gamma chain (gammac) by calpain.

The small subunit of calpain, a calcium-dependent cysteine protease, was found to interact with the cytoplasmic domain of the common cytokine receptor gamma chain (gammac) in a yeast two-hybrid interaction trap assay. This interaction was functional as demonstrated by the ability of calpain to cleave in vitro-translated wild-type gammac, but not gammac containing a mutation in the PEST (proline, glutamate, serine, and threonine) sequence in its cytoplasmic domain, as well as by the ability of endogenous calpain to mediate cleavage of gammac in a calcium-dependent fashion. In T cell receptor-stimulated murine thymocytes, calpain inhibitors decreased cleavage of gammac. Moreover, in single positive CD4(+) thymocytes, not only did a calpain inhibitor augment CD3-induced proliferation, but antibodies to gammac blocked this effect. Finally, treatment of cells with ionomycin could inhibit interleukin 2-induced STAT protein activation, but this inhibition could be reversed by calpain inhibitors. Together, these data suggest that calpain-mediated cleavage of gammac represents a mechanism by which gammac-dependent signaling can be controlled.

Do CTL kill target cells by inducing apoptosis?

Inhibitors of ICE-family proteases (caspases) block many examples of apoptotic cell death in vivo and in vitro, including multiple apoptotic stimuli for T lymphocytes. We have tested whether cell death induced by cytotoxic T lymphocytes was also blocked by caspase inhibitors. We found that the rapid apoptotic target cell death induced by Fas ligand-bearing CTL using the target Fas death pathway was efficiently blocked by caspase inhibitors. In contrast, target lysis induced by the CTL granule exocytosis pathway is not detectably blocked by such inhibitors, although the accompanying apoptotic nuclear damage is efficiently blocked. Thus caspase inhibitors prevent the hallmark phenotype of apoptosis without measurably affecting target cell death as evidenced by lysis.

Target cell lysis by CTL granule exocytosis is independent of ICE/Ced-3 family proteases.

Activation of ICE/Ced-3 family proteases (caspases) has been proposed to mediate both the granule exocytosis and Fas-Fas ligand pathways of rapid target cell death by cytotoxic T lymphocytes. In agreement with this model, two peptide fluoromethyl ketone caspase inhibitors and baculovirus p35 blocked apoptotic nuclear damage and target cell lysis by the CTL-mediated Fas-Fas ligand pathway. The peptide caspase inhibitors also blocked drug-induced apoptotic cell death in tumor cells. In contrast, the caspase inhibitors blocked CTL granule exocytosis-induced target apoptotic nuclear damage, but did not inhibit target lysis. These results are consistent with recent demonstrations that granzyme B can activate caspases leading to apoptotic nuclear damage, but show that target cell lysis by CTL granule exocytosis occurs by a caspase-independent pathway.

Apoptotic cell death and cytokine dysregulation in human immunodeficiency virus infection: pivotal factors in disease progression.

The progressive loss of CD4 T lymphocyte is patognomonic of Human Immunodeficiency Virus (HIV) infection and results in immunodeficiency and the appearance of acquired immunodeficiency syndrome (AIDS)-defining pathologies. Although a percentage of CD4 T lymphocytes is destroyed directly by HIV infection, a much higher proportion of lymphocytes remains uninfected and therefore must be destroyed by mechanisms not directly involving viral infection. One such mechanism is apoptotic T cell death (ATCD). ATCD in HIV infection has been shown to be: 1) secondary to cross-linking of CD4 by viral proteins; 2) mediated by both APO-1/Fas and lymphotoxin (LT); and 3) differentially modulated by type 1 and type 2 cytokines. We will briefly analyze the experimental evidences suggesting that ATCD contributes significantly to the immunopathogenesis of HIV/AIDS via depletion of CD4+ T cells.

Different interleukin-1 beta converting enzyme (ICE) family protease requirements for the apoptotic death of T lymphocytes triggered by diverse stimuli.

Two cell permeable peptide fluoromethyl ketone inhibitors of Interleukin-1 beta converting enzyme (ICE) family proteases were tested as inhibitors of apoptotic cell death of T lymphocytes at various stages of differentiation. The CPP-32-like protease activity in apoptotic cell lysates was blocked by both the ICE inhibitor Cbz-Val-Ala-Asp(OMe)-fluoromethyl ketone (ZVAD-FMK) as well as its truncated analog Boc-Asp(OMe)-fluoromethyl ketone (BD-FMK), which failed to block ICE. In vitro apoptotic death in murine thymocytes triggered by the independent agents dexamethasone, etoposide, radiation, anti-Fas, and anti-CD3 was blocked equally well by BD-FMK and ZVAD-FMK, but not by the control reagent Cbz-Phe-Ala-fluoromethyl ketone. In activated T cell blasts, while anti-CD3/ Fas-induced death was almost completely inhibited by both ZVAD-FMK, and BD-FMK, death induced by dexamethasone, etoposide, or irradiation was more sensitive to inhibition by BD-FMK. In the murine T cell line CTLL-2, apoptotic death induced by IL-2 withdrawal, etoposide, or dexamethasone was inhibited by BD-FMK, while ZVAD-FMK was without effect. These data indicate that ICE-family proteases comprise a common functional step in distinct T cell apoptotic death pathways, but suggest that different family members are likely to be critical in various differentiated T cell types, even when triggered by the same stimulus.

Role of reactive oxygen intermediates in TCR-induced death of T cell blasts and hybridomas.

The functional role of reactive oxygen intermediates (ROI) in activation-induced death of mature T lymphocytes and hybridomas was tested using antioxidants and inhibitors of enzymes that generate oxidants. These agents were shown to inhibit TCR-triggered death in a concentration-dependent manner, suggesting a possible role of ROI in this death. Since the TCR-induced death of both human T blasts and the murine T cell hybridoma 2B4 involve an initial step of TCR-induced Fas ligand (FasL) up-regulation followed by a lethal step induced by Fas cross-linking, both steps were examined separately for ROI dependence. The thiol antioxidants N-acetyl cysteine and glutathione blocked Fas-induced death triggered via cross-linking either by IgM anti-Fas or cell-bound FasL, while the other inhibitors of activation-induced death did not block this late lethal step. None of the agents used blocked early events after TCR ligation, as seen by the lack of inhibition of IL-2 secretion or CD69 up-regulation. However, the nonthiol agents that blocked activation-induced death all blocked FasL up-regulation induced by TCR signals in the hybridoma, as measured by a functional assay. Agents inhibiting FasL up-regulation also inhibited activation-induced ROI generation in the hybridoma, as detected by flow cytometry using dihydrorhodamine oxidation. Furthermore, a good correlation was found between the extent of ROI generation and functional FasL expression in 2B4 cells. Thus, while ROI do not appear to act as downstream mediators of apoptotic death induced by steroid or Fas cross-linking in T cells, they are generated by TCR signaling and appear to participate in FasL up-regulation.

Antigen-stimulated apoptotic T-cell death in HIV infection is selective for CD4+ T cells, modulated by cytokines and effected by lymphotoxin.

OBJECTIVE: To characterize the mechanism of in vitro antigen-induced apoptotic T-cell death in the peripheral blood mononuclear cells (PBMC) of HIV-1-infected individuals. DESIGN AND METHODS: PBMC from HIV-1 infected and uninfected individuals were unstimulated or stimulated with HIV-1 envelope synthetic peptides (Env) or influenza A virus to determine the extent of antigen-stimulated apoptotic T-cell death, whether this death was limited to the CD4+ subset, and the effects of cytokines on T-cell death. Death was assessed by apoptotic nuclear morphology after 7 days of culture by fluorescence microscopy using a DNA-specific dye. Transwell cultures and supernatant transfers were utilized to test whether a soluble factor produced by HIV-positive PBMC induced death of HIV-negative T cells. Exogenous cytokines [interleukin (IL)-12, interferon (IFN)-gamma, IL-4 and IL-10], as well as antibodies against endogenously produced cytokines (IL-4, IL-10, IL-12, and lymphotoxin) were tested for their ability to modulate death. RESULTS: Antigenic stimulation induced death in PBMC from HIV-positive donors, but not in PBMC from HIV-negative donors. Antigen-stimulated death was seen in CD4+ but not CD8+ T-cell subset from the HIV-positive patients. Apoptotic death was blocked by IL-12, IFN-gamma, anti-IL-4, anti-IL-10, and anti-lymphotoxin, but not by anti-IL-12. Transwell and supernatant transfer experiment indicated that antigen-stimulated HIV-positive PBMC produced a factor that killed T-cell blasts. The factor was inhibited by anti-lymphotoxin, but not by anti-IL-10. CONCLUSIONS: Stimulation of HIV-positive PBMC with CD4-dependent antigens results in selective death of CD4+ T cells that is modulated by cytokines. Our results suggest that apoptotic death is not limited to HIV-infected or HIV-specific T cells, but occurs in bystander cells. Lymphotoxin is a mediator of antigen-stimulated T-cell death in this in vitro model.