Caspase-independent cell death by Fas ligation in human thymus-derived T cell line, HPB-ALL cells

In HPB-ALL cells, a human thymus-derived T-cell line, Fas (CD95)-mediated cell death was inhibited by about only 50% as a result of treatment with an amount of benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)-CH(2)F (zVAD-fmk) sufficient to block the caspase activity. Fas-mediated caspase-independent cell death was not observed in other lymphoblast cell lines or mouse activated splenocytes, but this type of cell death was observed in mouse and rat thymocytes, the same as for HPB-ALL cells. This suggests that Fas-mediated caspase-independent cell death is a common feature in thymocytes. The signaling pathway of caspase-independent cell death has not yet been fully elucidated. In HPB-ALL cells, DNA fragmentation, one of the features of apoptotic cells, did not occur in the caspase-independent cell death after Fas ligation. On the other hand, this type of cell death and the surface exposure of phosphatidylserine were recovered by pretreatment with geldanamycin, which brought about a decrease in receptor interacting protein (RIP) kinase expression. These results suggested that HPB-ALL cells have a caspase-independent RIP kinasedependent pathway for Fas ligation.     

Identification of CLEC12B, an inhibitory receptor on myeloid cells

Activation of immune cells has to be tightly controlled to prevent detrimental hyperactivation. In this regulatory process molecules of the C-type lectin-like family play a central role. Here we describe a new member of this family, CLEC12B. The extracellular domain of CLEC12B shows considerable homology to the activating natural killer cell receptor NKG2D, but unlike NKG2D, CLEC12B contains an immunoreceptor tyrosine-based inhibition motif in its intracellular domain. Despite the homology, CLEC12B does not appear to bind NKG2D ligands and therefore does not represent the inhibitory counterpart of NKG2D. However, CLEC12B has the ability to counteract NKG2D-mediated signaling, and we show that this function is dependent on the immunoreceptor tyrosine-based inhibition motif and the recruitment of the phosphatases SHP-1 and SHP-2. Using monoclonal anti-CLEC12B antibodies we found de novo expression of this receptor on in vitro generated human macrophages and on the human myelo-monocytic cell line U937 upon phorbol 12-myristate 13-acetate treatment, suggesting that this receptor plays a role in myeloid cell function.     

The inhibitory receptor IRp60 (CD300a) is expressed and functional on human mast cells

Mast cell-mediated responses are likely to be regulated by the cross talk between activatory and inhibitory signals. We have screened human cord blood mast cells for recently characterized inhibitory receptors expressed on NK cells. We found that IRp60, an Ig superfamily member, is expressed on human mast cells. On NK cells, IRp60 cross-linking leads to the inhibition of cytotoxic activity vs target cells in vitro. IRp60 is constitutively expressed on mast cells but is down-regulated in vitro by the eosinophil proteins major basic protein and eosinophil-derived neurotoxin. An immune complex-mediated cross-linking of IRp60 led to inhibition of IgE-induced degranulation and stem cell factor-mediated survival via a mechanism involving tyrosine phosphorylation, phosphatase recruitment, and termination of cellular calcium influx. To evaluate the role of IRp60 in regulation of allergic responses in vivo, a murine model of allergic peritonitis was used in which the murine homolog of IRp60, LMIR1, was neutralized in BALB/c mice by mAbs. This neutralization led to a significantly augmented release of inflammatory mediators and eosinophilic infiltration. These data demonstrate a novel pathway for the regulation of human mast cell function and allergic responses, indicating IRp60 as a candidate target for future treatment of allergic and mast cell-associated diseases.     

KIR2DL4 (CD158d), an NK cell-activating receptor with inhibitory potential

KIR2DL4 (CD158d) is an unusual member of the killer cell Ig-like receptor family expressed in all NK cells and some T cells. KIR2DL4 activates the cytotoxicity of NK cells, despite the presence of an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic tail. The role of this ITIM on the activating function of KIR2DL4, and whether it can provide inhibitory signals, is not known. Mutated forms of KIR2DL4 were engineered that lacked either the tyrosine in the ITIM or an arginine-tyrosine motif in the transmembrane region that is required for the activation signal. The activity of the mutated KIR2DL4 molecules was tested in a redirected lysis assay. The ITIM was not necessary for activation of lysis by KIR2DL4. The activation signal of KIR2DL4 was sensitive to inhibition by another ITIM-containing receptor. The activation-deficient mutant of KIR2DL4 inhibited the signal delivered by the activating receptor CD16. In pull-down experiments with GST fusion proteins, the tyrosine-phosphorylated cytoplasmic tail of KIR2DL4 bound the Src homology 2-containing phosphatases 1 and 2, as did the tail of the inhibitory receptor KIR2DL1. Therefore, KIR2DL4 has inhibitory potential in addition to its activating function.     

Caspase-independent pathways of hair cell death induced by kanamycin in vivo

Cochlear and vestibular sensory cells undergo apoptosis when exposed to aminoglycoside antibiotics in organ culture, but mechanisms of chronic drug-induced hair cell loss in vivo are unclear. We investigated cell death pathways in a mouse model of progressive kanamycin-induced hair cell loss. Hair cell nuclei showed both apoptotic- and necrotic-like appearances but markers for classic apoptotic pathways (cytochrome c, caspase-9, caspase-3, JNK, TUNEL) were absent. In contrast, drug treatment caused EndoG translocation, activation of mu-calpain, and both the synthesis and activation of cathepsin D. Poly (ADP-ribose) polymerase 1 (PARP1) was decreased, but a caspase-derived 89 kDa PARP1 fragment was not present. The mRNA level of PARP1 remained unchanged. Thus, chronic administration of aminoglycosides causes multiple forms of cell death, without a major contribution by classic apoptosis. These results provide a better understanding of the toxic effects of aminoglycosides and are relevant to design protection from aminoglycoside-induced hearing loss.     

Caspase-independent cell death and mitochondrial disruptions observed in the Apaf1-deficient cells

Apaf1 is a critical molecule in the mitochondria-dependent apoptotic pathway. Here we show that Apaf1-deficient embryonic fibroblasts died at a later phase of apoptotic induction, although these cells were resistant to various apoptotic stimulants at an early phase. Neither caspase 3 activation nor nuclear condensation was observed during this cell death of Apaf1-deficient cells. Electron microscopic examination revealed that death in response to apoptotic stimulation resembled necrosis in that nuclei were round and swollen with low electron density. Necrosis-like cell death was also observed in wild-type cells treated with z-VAD-fmk. Mitochondria were not only morphologically abnormal but functionally affected, since mitochondrial transmembrane potential (DeltaPsim) was lost even in cells with intact plasma membrane integrity. These mitochondrial alterations were also observed in the wild-type cells dying of apoptosis. Combined, these data suggest that cells without caspase activation, such as Apaf1-deficient cells or cells treated with caspase inhibitors, die of necrosis-like cell death with mitochondrial damage in response to "apoptotic stimulation."     

Caspase-independent cell death does not elicit a proliferative response in melanoma cancer cells

Background

Apoptosis, the most well-known type of programmed cell death, can induce in a paracrine manner a proliferative response in neighboring surviving cells called apoptosis-induced proliferation (AiP). While having obvious benefits when triggered in developmental processes, AiP is a serious obstacle in cancer therapy, where apoptosis is frequently induced by chemotherapy. Therefore, in this study, we evaluated the capacity of an alternative type of cell death, called caspase-independent cell death, to promote proliferation.

Results

Using a novel in vitro isogenic cellular model to trigger either apoptosis or caspase-independent cell death, we found that the later has no obvious compensatory proliferation effects on neighboring cells.

Conclusions

This study enforces the idea that alternative types of cell death such as caspase-independent cell death could be considered to replace apoptosis in the context of cancer treatment.

     

T cell receptor/CD3-signaling induces death by apoptosis in human T cell receptor gamma delta + T cells.

mAb directed against the TCR/CD3 complex activate resting T cells. However, TCR/CD3 signaling induces death by apoptosis in immature (CD4+CD8+) murine thymocytes and certain transformed leukemic T cell lines. Here we show that anti-TCR and anti-CD3 mAb induce growth arrest of cloned TCR-gamma delta + T cells in the presence of IL-2. In the absence of exogenous IL-2, however, the very same anti-TCR/CD3 mAb stimulated gamma delta (+)-clones to proliferation and IL-2 production. In the presence of exogenous IL-2, anti-TCR/CD3 mAb induced the degradation of DNA into oligosomal bands of approximately 200 bp length in cloned gamma delta + T cells. This pattern of DNA fragmentation is characteristic for the programmed cell death termed apoptosis. These results demonstrate that TCR/CD3 signaling can induce cell death in cloned gamma delta + T cells. In addition, this report is the first to show that apoptosis triggered by TCR/CD3 signaling is not restricted to CD4+CD8+ immature thymocytes and transformed leukemic T cell lines but can be also observed with IL-2-dependent normal (i.e., TCR-gamma delta +) T cells.     

Superantigen-induced CD4 T cell tolerance mediated by myeloid cells and IFN-gamma

We have previously shown that systemic staphylococcal enterotoxin A (SEA) injections cause CD4 T cells in TCR-transgenic mice to become tolerant to subsequent ex vivo restimulation. An active IFN-gamma-dependent mechanism of suppression was responsible for the apparent unresponsiveness of the CD4 T cells. In this study, we analyze the response of CD4 T cells isolated throughout the first 10 days of the in vivo response to injected SEA. We show that CD4 T cells isolated at the peak of the in vivo response undergo very little activation-induced cell death after sterile FACS sorting or restimulation in the presence of neutralizing Abs to IFN-gamma. We also show that the IFN-gamma-dependent tolerance develops soon after SEA injection in the spleens of both normal and TCR-transgenic mice. This suppression is dependent upon myeloid cells from the SEA-treated mice and is optimal when inducible NO synthase activity and reactive oxygen intermediates are both present. The data indicate that IFN-gamma, myeloid cells, and a combination of NO and reactive oxygen intermediates all contribute to a common pathway of T cell death that targets activated or responding CD4 T cells. Sorted Gr-1(+) cells from SEA-treated mice also directly suppress the response of naive CD4 T cells in mixed cultures, indicating that this tolerance mechanism may play a role in down-regulating other vigorous immune responses.     

Regulation of myeloid cell function through the CD200 receptor

Myeloid cells play pivotal roles in chronic inflammatory diseases through their broad proinflammatory, destructive, and remodeling capacities. CD200 is widely expressed on a variety of cell types, while the recently identified CD200R is expressed on myeloid cells and T cells. CD200 deletion in vivo results in myeloid cell dysregulation and enhanced susceptibility to autoimmune inflammation, suggesting that the CD200-CD200R interaction is involved in immune suppression. We demonstrate in this study that CD200R agonists suppress mouse and human myeloid cell function in vitro, and also define a dose relationship between receptor expression and cellular inhibition. IFN-gamma- and IL-17-stimulated cytokine secretion from mouse peritoneal macrophages was inhibited by CD200R engagement. Inhibitory effects were not universal, as LPS-stimulated responses were unaffected. Inhibition of U937 cell cytokine production correlated with CD200R expression levels, and inhibition was only observed in low CD200R expressing cells, if the CD200R agonists were further cross-linked. Tetanus toxoid-induced human PBMC IL-5 and IL-13 secretion was inhibited by CD200R agonists. This inhibition was dependent upon cross-linking the CD200R on monocytes, but not on cross-linking the CD200R on CD4+ T cells. In all, we provide direct evidence that the CD200-CD200R interaction controls monocyte/macrophage function in both murine and human systems, further supporting the potential clinical application of CD200R agonists for the treatment of chronic inflammatory diseases.     

Molecular mechanism of the activation-induced cell death inhibition mediated by a p70 inhibitory killer cell Ig-like receptor in Jurkat T cells

In this study we investigated the molecular mechanism of the activation-induced cell death (AICD) inhibition mediated by a p70 inhibitory killer cell Ig-like receptor (KIR3DL1, also called NKB1) in Jurkat T cells. Using stable Jurkat transfectants that express KIR or CD8-KIR fusion proteins we have shown for the first time that KIR inhibits, in a ligation-independent manner, the AICD induced by PHA, PMA/ionomycin, or anti-CD3 Ab. The AICD inhibition mediated by KIR appears to result from the blockade of Fas ligand induction upon activation of the Jurkat transfectants. Moreover, the membrane-proximal 20 aa of the KIR cytoplasmic tail were determined to play a crucial role in this process. Since the membrane-proximal portion of the KIR cytoplasmic tail contains a putative protein kinase C (PKC) substrate site, we investigated the molecular interaction between KIR and PKC. Immunoprecipitation analysis demonstrated that KIR constitutively bound both to PKCalpha, a conventional Ca(2+)-dependent PKC, and to PKCtheta, a novel Ca(2+)-independent PKC. Furthermore, an in vitro kinase assay revealed that PKC activation was blocked after PHA stimulation in Jurkat transfectants expressing KIR. These observations were supported by the finding that a recombinant KIR cytoplasmic tail also appeared to inhibit PKCalpha activation in vitro. Taken together these data strongly suggest that KIR inhibits the AICD of T cells by blocking Fas ligand induction upon stimulation, in a process that seems to be accomplished by PKC recruitment to the membrane-proximal PKC binding site and subsequent inhibition of PKC activation against the activating stimuli.     

CD300 heterocomplexes, a new and family-restricted mechanism for myeloid cell signaling regulation

The CD300 family of myeloid immunoglobulin receptors includes activating (CD300b, CD300e) and inhibitory members (CD300a, CD300f), as well as molecules of uncertain function presenting a negative charge within their transmembrane domain (CD300c, CD300d). In this paper, we establish that CD300c is a functional immune receptor able to deliver activating signals upon ligation in RBL-2H3 mast cells. CD300c signaling is partially mediated by a direct association with the immune receptor tyrosine-based activation motif-bearing adaptor FcεRγ. The existence of complementary transmembrane-charged residues in certain CD300 receptors suggested the formation of heterodimers within this family. Indeed, we proved the interaction between CD300b and CD300c in transfected COS-7 cells and demonstrated that it has important functional consequences. Unexpectedly, dimmer formation was dependent on the immunoglobulin domains rather than the charged transmembrane residues. Concordantly, all CD300 members were found to interact with each other, even with themselves, forming both homo- and heterodimers. We found that the combination of CD300 receptors in a complex differentially modulates the signaling outcome, strongly suggesting a new mechanism by which CD300 complexes could regulate the activation of myeloid cells upon interaction with their natural ligands.     

Leukemia inhibitory factor (LIF), cardiotrophin-1, and oncostatin M share structural binding determinants in the immunoglobulin-like domain of LIF receptor

Leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), and oncostatin M (OSM) are four helix bundle cytokines acting through a common heterodimeric receptor composed of gp130 and LIF receptor (LIFR). Binding to LIFR occurs through a binding site characterized by an FXXK motif located at the N terminus of helix D (site III). The immunoglobulin (Ig)-like domain of LIFR was modeled, and the physico-chemical properties of its Connolly surface were analyzed. This analysis revealed an area displaying properties complementary to those of the LIF site III. Two residues of the Ig-like domain of LIFR, Asp214 and Phe284, formed a mirror image of the FXXK motif. Engineered LIFR mutants in which either or both of these two residues were mutated to alanine were transfected in Ba/F3 cells already containing gp130. The F284A mutation impaired the biological response induced by LIF and CT-1, whereas the response to OSM remained unchanged. The Asp214 mutation did not alter the functional responses. The D214A/F284A double mutation, however, totally impaired cellular proliferation to LIF and CT-1 and partially impaired OSM-induced proliferation with a 20-fold increase in EC50. These results were corroborated by the analysis of STAT3 phosphorylation and Scatchard analysis of cytokine binding to Ba/F3 cells. Molecular modeling of the complex of LIF with the Ig-like domain of LIFR provides a clue for the superadditivity of the D214A/F284A double mutation. Our results indicate that LIF, CT-1, and OSM share an overlapping binding site located in the Ig-like domain of LIFR. The different behaviors of LIF and CT-1, on one side, and of OSM, on the other side, can be related to the different affinity of their site III for LIFR.     

Suppression of androgen receptor signaling in prostate cancer cells by an inhibitory receptor variant

There is increasing evidence that sensitization of the androgen receptor (AR) signaling pathway contributes to the failure of androgen ablation therapy for prostate cancer, and that direct targeting of the AR may be a useful therapeutic approach. To better understand how AR function could be abrogated in prostate cancer cells, we have developed a series of putative dominant-negative variants of the human AR, containing deletions or mutations in activation functions AF-1, AF-5, and/or AF-2. One construct, AR inhibitor (ARi)-410, containing a deletion of AF-1 and part of AF-5 of the AR, had no intrinsic transactivation activity but inhibited wild-type AR (wtAR) in a ligand-dependent manner by at least 95% when transfected at a 4:1 molar ratio. ARi-410 was an equally potent inhibitor of gain-of-function AR variants. Ectopic expression of ARi-410 inhibited the proliferation of AR-positive LNCaP cells, but not AR-negative PC-3 cells. Whereas ARi-410 also marginally inhibited progesterone receptor activity, this was far less pronounced than the effect on AR (50% vs. 95% maximal inhibition, respectively), and there was no inhibition of either vitamin D or estrogen receptor activity. In the presence of ligand, ARi-410 interacted with wtAR, and both receptors translocated into the nucleus. Whereas the amino-carboxy terminal interaction was not necessary for optimal dominant-negative activity, disruption of dimerization through the ligand binding domain reduced the efficacy of ARi-410. In addition, although inhibition of AR function by ARi-410 was not dependent on DNA binding, the DNA binding domain was required for dominant-negative activity. Taken together, our results suggest that interaction between ARi-410 and the endogenous AR in prostate cancer cells, potentially through the DNA binding and ligand binding domains, results in a functionally significant reduction in AR signaling and AR-dependent cell growth.     

Adhesion to target cells is disrupted by the killer cell inhibitory receptor

Killer cell immunoglobulin-like receptors (KIR) inhibit the cytotoxic activity of natural killer (NK) cells by recruitment of the tyrosine phosphatase SHP-1 to immunoreceptor tyrosine-based inhibition motif (ITIM) sequences in the KIR cytoplasmic tail [1]. The precise steps in the NK activation pathway that are inhibited by KIR are yet to be defined. Here, we have studied whether the initial step of adhesion molecule LFA-1-dependent adhesion to target cells was altered by the inhibitory signal. Using stable expression of an HLA-C-specific KIR in the NK cell line YTS [2] and a two-color flow cytometry assay for conjugate formation, we show that adhesion to a target cell expressing cognate HLA-C was disrupted by KIR engagement. Conjugate formation was abruptly interrupted by KIR within less than 5 minutes. Inhibition of adhesion to target cells was mediated by a chimeric KIR molecule carrying catalytically active SHP-1 in place of its cytoplasmic tail. These results suggest that other ITIM-bearing receptors, many of which have no known function, may regulate adhesion in a wide variety of cell types.     

Mast cell death induced by 24(S),25-epoxycholesterol

Mast cell is one of the central effectors in inflammatory responses. Recent studies suggest that a promising therapeutic approach for various inflammatory immune diseases, including rheumatoid arthritis, multiple sclerosis, and type I allergies, is to inhibit mast cell growth and/or survival. Studies also indicate that a balanced lipid metabolism is crucial for regulating the life span of cells. Oxysterol is a well-known regulator of lipid metabolism and has diverse functions, such as inhibition of the mevalonate isoprenoid pathway, efflux of free cholesterols, and synthesis of cholesterol esters. Here, we show that 24(S),25-epoxycholesterol, a representative endogenous oxysterol, induces apoptosis in bone marrow-derived murine mast cells. Furthermore, we have revealed, for the first time, that the accumulation of neutral lipids catalyzed by acyl-CoA:cholesterol acyltransferase in the cells was involved in induction of mast cell apoptosis. Our present findings confer new insights into the roles of lipid metabolism during oxysterol-mediated mast cell apoptosis.     

Tumor-associated CD8+ T cell tolerance induced by bone marrow-derived immature myeloid cells

T cell tolerance is a critical element of tumor escape. However, the mechanism of tumor-associated T cell tolerance remains unresolved. Using an experimental system utilizing the adoptive transfer of transgenic T cells into naive recipients, we found that the population of Gr-1+ immature myeloid cells (ImC) from tumor-bearing mice was able to induce CD8+ T cell tolerance. These ImC accumulate in large numbers in spleens, lymph nodes, and tumor tissues of tumor-bearing mice and are comprised of precursors of myeloid cells. Neither ImC from control mice nor progeny of tumor-derived ImC, including tumor-derived CD11c+ dendritic cells, were able to render T cells nonresponsive. ImC are able to take up soluble protein in vivo, process it, and present antigenic epitopes on their surface and induce Ag-specific T cell anergy. Thus, this is a first demonstration that in tumor-bearing mice CD8+ T cell tolerance is induced primarily by ImC that may have direct implications for cancer immunotherapy.     

Regulation of antiviral CD8+ T cells by inhibitory natural killer cell receptors

Recent evidence indicates that CD8(+) T cells express natural killer cell receptors that constrain the range and magnitude of their activities. For virus-specific CD8(+) T cells, upregulation of these receptors serves to control infection, while concurrently minimizing bystander pathology. Dysregulated expression of these receptors, however, may foster the establishment of persistent virus infection.     

Autophagic cell death induced by TrkA receptor activation in human glioblastoma cells

The neurotrophin receptor tropomyosin-related kinase A (TrkA) and its ligand nerve growth factor (NGF) are expressed in astrocytomas, and an inverse association of TrkA expression with malignancy grade was described. We hypothesized that TrkA expression might confer a growth disadvantage to glioblastoma cells. To analyze TrkA function and signaling, we transfected human TrkA cDNA into the human glioblastoma cell line G55. We obtained three stable clones, all of which responded with striking cytoplasmic vacuolation and subsequent cell death to NGF. Analyzing the mechanism of cell death, we could exclude apoptosis and cellular senescence. Instead, we identified several indications of autophagy: electron microscopy showed typical autophagic vacuoles; acridine orange staining revealed acidic vesicular organelles; acidification of acidic vesicular organelles was prevented using bafilomycin A1; cells displayed arrest in G2/M; increased processing of LC3 occurred; vacuolation was prevented by the autophagy inhibitor 3-methyladenine; no caspase activation was detected. We further found that both activation of ERK and c-Jun N-terminal kinase but not p38 were involved in autophagic vacuolation. To conclude, we identified autophagy as a novel mechanism of NGF-induced cell death. Our findings suggest that TrkA activation in human glioblastomas might be beneficial therapeutically, especially as several of the currently used chemotherapeutics also induce autophagic cell death.     

CD95 death receptor and epidermal growth factor receptor (EGFR) in liver cell apoptosis and regeneration

Recent evidence suggests that signaling pathways towards cell proliferation and cell death are much more interconnected than previously thought. Whereas not only death receptors such as CD95 (Fas, APO-1) can couple to both, cell death and proliferation, also growth factor receptors such as the epidermal growth factor receptor (EGFR) are involved in these opposing kinds of cell fate. EGFR is briefly discussed as a growth factor receptor involved in liver cell proliferation during liver regeneration. Then the role of EGFR in activating CD95 death receptor in liver parenchymal cells (PC) and hepatic stellate cells (HSC), which represent a liver stem/progenitor cell compartment, is described summarizing different ways of CD95- and EGFR-dependent signaling in the liver. Here, depending on the hepatic cell type (PC vs. HSC) and the respective signaling context (sustained vs. transient JNK activation) CD95-/EGFR-mediated signaling ends up in either liver cell apoptosis or cell proliferation.