Cartilage tissue engineering relies on in vitro expansion of primary chondrocytes. Monolayer is the chosen culture model for chondrocyte expansion because in this system the proliferative capacity of chondrocytes is substantially higher compared to non-adherent systems. However, human articular chondrocytes (HACs) cultured as monolayers undergo changes in phenotype and gene expression known as "dedifferentiation." To gain a better understanding of the cellular mechanisms involved in the dedifferentiation process, our research focused on the characterization of the surface molecule phenotype of HACs in monolayer culture. Adult HACs were isolated by enzymatic digestion of cartilage samples obtained post-mortem. HACs cultured in monolayer for different time periods were analyzed by flow cytometry for the expression of cell surface markers with a panel of 52 antibodies. Our results show that HACs express surface molecules belonging to different categories: integrins and other adhesion molecules (CD49a, CD49b, CD49c, CD49e, CD49f, CD51/61, CD54, CD106, CD166, CD58, CD44), tetraspanins (CD9, CD63, CD81, CD82, CD151), receptors (CD105, CD119, CD130, CD140a, CD221, CD95, CD120a, CD71, CD14), ectoenzymes (CD10, CD26), and other surface molecules (CD90, CD99). Moreover, differential expression of certain markers in monolayer culture was identified. Up-regulation of markers on HACs regarded as distinctive for mesenchymal stem cells (CD10, CD90, CD105, CD166) during monolayer culture suggested that dedifferentiation leads to reversion to a primitive phenotype. This study contributes to the definition of HAC phenotype, and provides new potential markers to characterize chondrocyte differentiation stage in the context of tissue engineering applications.

Stimulation of previously activated T cells through the antigen receptor can result in the apoptotic death of the responding cell, a process referred to as activation-induced cell death (AICD). This process appears to involve Fas (CD95) and its ligand (Fas-L). The distribution of Fas and Fas-L on various T cell subsets has not been extensively characterized. We have therefore analyzed cells committed to a Th1- or Th2-type differentiation pattern for the expression and function of Fas-L. Using both a sensitive bioassay and flow cytometry, we demonstrate that cloned Th1 cells express high levels of Fas-L, whereas cloned Th2 cells express only low levels. The expression of Fas-L by Th1 and Th2 cells correlates with the relative abilities of these two cell types to undergo AICD. Whereas AICD is readily observed in cultures of cloned Th1, but not Th2 cells, Th2 cells are capable of undergoing apoptosis in the presence of Th1 cells expressing Fas-L. The ability of T cells to undergo AICD appears to be unrelated to the presence of various cytokines. Thus, the Fas/Fas-L pathway appears to be critical for the induction of AICD and this pathway is differentially regulated in cells committed to either Th1 or Th2 differentiation.

Anticancer agents have been shown to trigger apoptosis in chemosensitive tumors such as neuroblastomas. We previously identified activation of the CD95 system as one of the key mechanisms for doxorubicin-induced apoptosis in leukemic T cells. Here, we report that therapeutic concentrations of doxorubicin, cisplatinum, and VP-16 led to induction of CD95 receptor and CD95 ligand (CD95-L) that mediated cell death in chemosensitive neuroblastoma cells. Using F(ab')2 anti-CD95 antibody fragments to interfere with CD95-L-receptor interaction markedly reduced apoptosis induced by those drugs in vitro. Cyclosporin A inhibited induction of CD95 mRNA and CD95-L mRNA and blocked drug-mediated apoptosis. Drug-induced apoptosis involved activation of caspases (interleukin 1beta-converting enzyme/Ced-3-like proteases) and processing of the prototype caspase substrate PARP and was completely blocked by benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a peptide inhibitor of caspases. In addition, neuroblastoma cells that were resistant to CD95-triggered apoptosis also displayed cross-resistance to chemotherapeutic agents. These data provide new clues for understanding the molecular requirements for drug-induced apoptosis in chemosensitive neuroblastoma cells by demonstrating that cell death was mediated via the CD95-L-receptor system and may open new avenues for targeting drug resistance of neuroblastoma.

The antiapoptotic protein cellular FLICE (Fas-associated death domain-like IL-1beta-converting enzyme) inhibitory protein (cFLIP) protects cells from CD95(APO-1/Fas)-induced apoptosis in vitro and was found to be overexpressed in human melanomas. However, cytotoxic T cell-induced apoptosis, which is critically involved in tumor control in vivo, is not inhibited by cFLIP in vitro, as only CD95- and not perforin-dependent lysis is affected. This calls into question whether cFLIP is sufficient to allow escape from T cell-dependent immunity. Using two murine tumors, we directly demonstrate that cFLIP does result in escape from T cell immunity in vivo. Moreover, tumor cells are selected in vivo for elevated cFLIP expression. Therefore, our data indicate that CD95-dependent apoptosis constitutes a more prominent mechanism for tumor clearance than has so far been anticipated and that blockade of this pathway can result in tumor escape even when the perforin pathway is operational.

Mesenchymal stem cells (MSC) are part of the bone marrow that provides signals supporting survival and growth of bystander hematopoietic stem cells (HSC). MSC modulate also the immune response, as they inhibit proliferation of lymphocytes. In order to investigate whether MSC can support survival of T cells, we investigated MSC capacity of rescuing T lymphocytes from cell death induced by different mechanisms. We observed that MSC prolong survival of unstimulated T cells and apoptosis-prone thymocytes cultured under starving conditions. MSC rescued T cells from activation induced cell death (AICD) by downregulation of Fas receptor and Fas ligand on T cell surface and inhibition of endogenous proteases involved in cell death. MSC dampened also Fas receptor mediated apoptosis of CD95 expressing Jurkat leukemic T cells. In contrast, rescue from AICD was not associated with a significant change of Bcl-2, an inhibitor of apoptosis induced by cell stress. Accordingly, MSC exhibited a minimal capacity of rescuing Jurkat cells from chemically induced apoptosis, a process disrupting the mitochondrial membrane potential regulated by Bcl-2. These results suggest that MSC interfere with the Fas receptor regulated process of programmed cell death. Overall, MSC can inhibit proliferation of activated T cells while supporting their survival in a quiescent state, providing a model of their activity inside the HSC niche. Disclosure of potential conflicts of interest is found at the end of this article.

Pseudomonas aeruginosa causes severe infections, particularly of the lung, that are life threatening. Here, we show that P. aeruginosa infection induces apoptosis of lung epithelial cells by activation of the endogenous CD95/CD95 ligand system. Deficiency of CD95 or CD95 ligand on epithelial cells prevented apoptosis of lung epithelial cells in vivo as well as in vitro. The importance of CD95/CD95 ligand-mediated lung epithelial cell apoptosis was demonstrated by the rapid development of sepsis in CD95- or CD95 ligand-deficient mice, but not in normal mice, after P. aeruginosa infection.

Fas/APO-1(CD95) ligation activates programmed cell death, a cellular process that plays an important role in the maturation of the host immune response. We show that activation of a specific MAP kinase kinase (MKK), MKK6b, is necessary and sufficient for Fas-induced apoptosis of Jurkat T cells. MKK6b activation occurs downstream of an interleukin-1 converting enzyme-like (ICE-like) protease(s), while execution of the apoptotic pathway by MKK6b requires both ICE- and CPP32-like proteases. Surprisingly, the p38 MAP kinase protein, a known substrate of MKK6b, does not participate in Fas/MKK6b-mediated apoptosis. These findings indicate a divergence of the MKK6b signaling pathways, one of which activates p38 and leads to regulation of gene expression, and one of which activates the ICE/Ced-3 family of proteases and leads to cell death. These studies represent a demonstration of an apoptotic pathway that is comprised of both the ICE/Ced-3 family of proteases and MAP kinase kinase 6.

Phosphatidylserine (PS), a lipid normally confined to the inner leaflet of the plasma membrane, is exported to the outer plasma membrane leaflet during apoptosis to serve as a trigger for recognition of apoptotic cells by phagocytes. The mechanism of PS export during apoptosis is not known nor is it clear whether the nuclear changes that typify apoptosis contribute in any way to this event. Here, we demonstrate that ligation of the CD95 (Fas/APO-1) molecule on Jurkat cytoplasts induces dramatic PS externalization similar to that observed during apoptosis of intact cells. Apoptosis of both cells and cytoplasts was associated with proteolytic processing of CPP32, a member of the interleukin-1beta converting enzyme (ICE)/CED-3 protease family, to its active form. Fodrin, a component of the cortical cytoskeleton, also underwent proteolytic cleavage during apoptosis of both cytoplasts and intact cells. Strikingly, CPP32 activation, fodrin proteolysis, and PS externalization were all inhibited in the presence of peptide inhibitors of ICE/CED-3 family proteases. These data provide strong support for the notion that the cell death machinery is extranuclear and is likely to be comprised of one or more members of the ICE/CED-3 family and that activation of this machinery does not require nuclear participation.

Regardless of whether apoptosis or necrosis are elicited by toxicants or by pathophysiological conditions they are considered conceptually distinct forms of cell death. Nevertheless, there is increasing evidence that classical apoptosis and necrosis represent only the extreme ends of a wide range of possible morphological and biochemical deaths. The two classical types of demise can occur simultaneously in tissues or cell cultures exposed to the same stimulus and often, the intensity of the same initial insult decides the prevalence of either apoptosis or necrosis. The execution of the death program seems to involve a relatively limited number of pathways. In many instances, their ordered execution results in characteristic morphological and biochemical changes termed apoptosis. However, some subroutines of the degradation program may not be active in all cases of cell death. Then, the morphological appearance of dying cells and some of their biochemical alterations differ from those of classical apoptosis. We have recently shown that intracellular energy levels and mitochondrial function are rapidly compromised in necrosis, but not in apoptosis of neuronal cells. Then we went on to show that pre-empting human T cells of ATP switches the type of demise caused by two classic apoptotic triggers (staurosporin and CD95 stimulation) from apoptosis to necrosis. Conditions of controlled intracellular ATP depletion, which was obtained by blocking mitochondrial and/or glycolytic ATP generation were used in combination with repletion of the cytosolic ATP pool with glucose to redirect the death program towards apoptosis or necrosis.

Optimal immunity to Mycobacterium tuberculosis (Mtb) infection requires CD8(+) T cells, and several current Mtb vaccine candidates are being engineered to elicit enhanced CD8(+) T cell responses. However, the function of these T cells and the mechanism by which they provide protection is still unknown. We have previously shown that CD8(+) T cells specific for the mycobacterial Ags CFP10 and TB10.4 accumulate in the lungs of mice following Mtb infection and have cytolytic activity in vivo. In this study, we determine which cytolytic pathways are used by these CD8(+) T cells during Mtb infection. We find that Mtb-specific CD8(+) T cells lacking perforin have reduced cytolytic capacity in vivo. In the absence of perforin, the residual cytolytic activity is CD95 and TNFR dependent. This is particularly true in Mtb-infected lung tissue where disruption of both perforin and CD95 eliminates target cell lysis. Moreover, adoptive transfer of immune CD8(+) T cells isolated from wild-type, but not perforin-deficient mice, protect recipient mice from Mtb infection. We conclude that CD8(+) T cells elicited following Mtb infection use several cytolytic pathways in a hierarchical and compensatory manner dominated by perforin-mediated cytolysis. Finally, although several cytolytic pathways are available, adoptively transferred Mtb-specific CD8(+) T cells require perforin-mediated cytolysis to protect animals from infection. These data show that CD8(+) T cell-mediated protection during Mtb infection requires more than the secretion of IFN-gamma and specifically defines the CD8(+) cytolytic mechanisms utilized and required in vivo.

Mesenchymal stem cells (MSCs) are known as a population of multi-potential cells able to proliferate and differentiate into multiple mesodermal tissues including bone, cartilage, muscle, ligament, tendon, fat and stroma. In this study human MSCs were successfully isolated from the umbilical cords. The research characteristics of these cells, e.g., morphologic appearance, surface antigens, growth curve, cytogenetic features, cell cycle, differentiation potential and gene expression were investigated. After 2weeks of incubation, fibroblast-like cells appeared to be dominant. During the second passage the cells presented a homogeneous population of spindle fibroblast-like cells. After more than 4months (approximately 26 passages), the cells continued to retain their characteristics. Flow cytometry analysis revealed that CD29, CD44, CD95, CD105 and HLA-I were expressed on the cell surface, but there was no expression of hematopoietic lineage markers, such as CD34, CD38, CD71 and HLA-DR. Chromosomal analysis showed the cells kept a normal karyotype. The cell cycle at the third passage showed the percentage of G(0)/G(1), G(2)/M and S phase were 88.86%, 5.69% and 5.45%, respectively. The assays in vitro demonstrated the cells exhibited multi-potential differentiation into osteogenic and adipogenic cells. Both BMI-1 and nucleostemin genes, expressed in adult MSCs from bone marrow, were also expressed in umbilical cord MSCs. Here we show that umbilical cords may be a novel alternative source of human MSCs for experimental and clinical applications.

Why HIV-specific CD8(+) T cells ultimately fail to clear or control HIV infection is not known. We show here that HIV-specific CD8(+) T cells exhibit increased sensitivity to CD95/Fas-induced apoptosis. This apoptosis is 3-fold higher compared to CMV-specific CD8(+) T cells from the same patients. HIV-specific CD8(+) T cells express the CD45RA(-)CD62L(-) but lack the CD45RA(+)CD62L(-) T cell effector memory (T(EM)) phenotype. This skewing is not found in CMV- and EBV-specific CD8(+) T cells in HIV-infected individuals. CD95/Fas-induced apoptosis is much higher in the CD45RA(-)CD62L(-) T(EM) cells. However, cytotoxicity and IFNgamma production by HIV-specific CD8(+) T cells is not impaired. Our data suggest that the survival and differentiation of HIV-specific CD8(+) T cells may be compromised by CD95/Fas apoptosis induced by FasL-expressing HIV-infected cells.

Genetic studies indicated that the Drosophila melanogaster protein REAPER (RPR) controls apoptosis during embryo development. Induction of RPR expression in Drosophila Schneider cells rapidly stimulated apoptosis. RPR-mediated apoptosis was blocked by N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), which suggests that an interleukin-1 beta converting enzyme (ICE)-like protease is required for RPR function. RPR-induced apoptosis was associated with increased ceramide production that was also blocked by Z-VAD-fmk, which suggests that ceramide generation requires an ICE-like protease as well. Thus, the intracellular RPR protein uses cell death signaling pathways similar to those used by the vertebrate transmembrane receptors Fas (CD95) and tumor necrosis factor receptor type 1.

Apoptosis is a fundamental biological process used to eliminate unwanted cells in a multicellular organism. An increasing number of regulatory proteins have been identified that either promote or inhibit apoptosis. For tumors to arise, apoptosis must be blocked in the transformed cells, for example by mutational overexpression of anti-apoptotic proteins, which represent attractive target proteins for molecular therapy strategies. In a functional yeast survival screen designed to select new anti-apoptotic mammalian genes, we have identified the chromosomal high-mobility group box-1 protein (HMGB1) as an inhibitor of yeast cell death induced by the pro-apoptotic Bcl-2 family member Bak. The C-terminal 33 amino acids of HMGB1 are dispensable for this inhibitory function. HMGB1 is also able to protect mammalian cells against different death stimuli including ultraviolet radiation, CD95-, TRAIL-, Casp-8-, and Bax-induced apoptosis. We found high HMGB1 protein levels in human primary breast carcinoma. Hmgb1 RNA levels are changing during different stages of mouse mammary gland development and are particularly low during lactation and involution. These data suggest that HMGB1 may participate in the regulation of mammary gland apoptosis and that its high expression level promotes tumor growth because of its anti-apoptotic properties.

We have shown previously that the death receptor CD95 could contribute to anticancer drug-induced apoptosis of colon cancer cells. In addition, anticancer drugs cooperate with CD95 cognate ligand or agonistic antibodies to trigger cancer cell apoptosis. In the present study, we show that the anticancer drug cisplatin induces clustering of CD95 at the surface of the human colon cancer cell line HT29, an event inhibited by the inhibitor of acid sphingomyelinase (aSMase) imipramine. The cholesterol sequestering agent nystatin also prevents cisplatin-induced CD95 clustering and decreases HT29 cell sensitivity to cisplatin-induced apoptosis and the synergy between cisplatin and anti-CD95 agonistic antibodies. CD95, together with the adaptor molecule Fas-associated death domain and procaspase-8, is redistributed into cholesterol- and sphingolipid-enriched cell fractions after cisplatin treatment, suggesting plasma membrane raft involvement. Interestingly, nystatin prevents the translocation of the aSMase to the extracellular surface of plasma membrane and the production of ceramide, suggesting that these early events require raft integrity. In addition, nystatin prevents cisplatin-induced transient increase in plasma membrane fluidity that could be required for CD95 translocation. Together, these results demonstrate that cisplatin activates aSMase and induces ceramide production, which triggers the redistribution of CD95 into the plasma membrane rafts. Such redistribution contributes to cell death and sensitizes tumor cells to CD95-mediated apoptosis.

Salinomycin is a polyether antibiotic isolated from Streptomyces albus that acts in different biological membranes as a ionophore with a preference for potassium. It is widely used as an anticoccidial drug in poultry and is fed to ruminants to improve nutrient absorption and feed efficiency. Salinomycin has recently been shown to selectively deplete human breast cancer stem cells from tumorspheres and to inhibit breast cancer growth and metastasis in mice. We show here that salinomycin induces massive apoptosis in human cancer cells of different origin, but not in normal cells such as human T lymphocytes. Moreover, salinomycin is able to induce apoptosis in cancer cells that exhibit resistance to apoptosis and anticancer agents by overexpression of Bcl-2, P-glycoprotein or 26S proteasomes with enhanced proteolytic activity. Salinomycin activates a distinct apoptotic pathway that is not accompanied by cell cycle arrest and that is independent of tumor suppressor protein p53, caspase activation, the CD95/CD95L system and the proteasome. Thus, salinomycin should be considered as a novel and effective anticancer agent that overcomes multiple mechanisms of apoptosis resistance in human cancer cells.

Activation-induced cell death is a process by which overactivated T cells are eliminated, thus preventing potential autoimmune attacks. Two known mediators of activation-induced cell death are Fas(<em>CD95</em>) ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL). We show here that upon mitogenic stimulation, bioactive FasL and APO2L are released from the T cell leukemia Jurkat and from normal human T cell blasts as intact, nonproteolyzed proteins associated with a particulate, ultracentrifugable fraction. We have characterized this fraction as microvesicles of 100-200 nm in diameter. These microvesicles are released from Jurkat and T cell blasts shortly (</=1 h) after PHA stimulation, well before the cell enters apoptosis. FasL- and APO2L-containing vesicles are also present in supernatants from PHA-activated fresh human PBMC. These observations provide the basis for a new and efficient mechanism for the rapid induction of autocrine or paracrine cell death during immune regulation.

Resveratrol, a plant antibiotic, has been found to have anticancer activity and was recently reported to induce apoptosis in the myeloid leukemia line HL60 by the CD95-CD95 ligand pathway. However, many acute lymphoblastic leukemias (ALLs), particularly of B-lineage, are resistant to CD95-mediated apoptosis. Using leukemia lines derived from patients with pro-B t(4;11), pre-B, and T-cell ALL, we show in this report that resveratrol induces extensive apoptotic cell death not only in CD95-sensitive leukemia lines, but also in B-lineage leukemic cells that are resistant to CD95-signaling. Multiple dose treatments of the leukemic cells with 50 microM resveratrol resulted in>>/=80% cell death with no statistically significant cytotoxicity against normal peripheral blood mononuclear cells under identical conditions. Resveratrol treatment did not increase CD95 expression or trigger sensitivity to CD95-mediated apoptosis in the ALL lines. Inhibition of CD95-signaling with a CD95-specific antagonistic antibody indicated that CD95-CD95 ligand interactions were not involved in initiating resveratrol-induced apoptosis. However, in each ALL line, resveratrol induced progressive loss of mitochondrial membrane potential as measured by the dual emission pattern of the mitochondria-selective dye JC-1. The broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone failed to block the depolarization of mitochondrial membranes induced by resveratrol, further indicating that resveratrol action was independent of upstream caspase-8 activation via receptor ligation. However, increases in caspase-9 activity ranged from 4- to 9-fold in the eight cell lines after treatment with resveratrol. Taken together, these results point to a general mechanism of apoptosis induction by resveratrol in ALL cells that involves a mitochondria/caspase-9-specific pathway for the activation of the caspase cascade and is independent of CD95-signaling.

OBJECTIVE

To determine phenotypic and functional abnormalities of blood B cell subsets in patients with systemic sclerosis (SSc).

METHODS

Cell surface marker expression was determined by flow cytometry. Spontaneous apoptosis was evaluated by annexin V expression with flow cytometric analysis. IgG production by isolated IgD- memory B cells was examined by enzyme-linked immunosorbent assay.

RESULTS

The numbers of blood CD27- naive B cells from SSc patients were increased compared with normal control cells, while memory B cells expressing medium levels of CD27 and plasmablasts expressing high levels of CD27 were reduced. In contrast, plasmablasts were the predominant population in patients with systemic lupus erythematosus (SLE). Memory B cells in SSc showed increased expression of activation markers, including CD80, CD86, and CD95, relative to normal controls. Consistent with CD95 up-regulation, SSc memory B cells exhibited augmented spontaneous apoptosis after 24-hour incubation; augmented apoptosis may explain the reduced memory B cell number. Nonetheless, isolated IgD- SSc memory B cells treated with stimuli had an enhanced ability to produce IgG. Furthermore, expression of CD19, a critical signal transduction molecule of B cells that regulates autoantibody production, was significantly increased in memory B cells as well as in naive B cells in SSc. In contrast, CD19 expression was decreased in SLE B cells.

CONCLUSIONS

SSc patients have distinct abnormalities of blood homeostasis and B cell compartments, characterized by expanded naive B cells and activated but diminished memory B cells. Our results suggest that CD19 overexpression in SSc memory B cells is related to their hyperreactivity.

Stimulation of the CD95/Fas/Apo-1 receptor leads to apoptosis through activation of the caspase family of cysteine proteases and disruption of the mitochondrial transmembrane potential (Deltapsim). We show that, in Jurkat human T cells and peripheral blood lymphocytes, Fas-induced apoptosis is preceded by 1) an increase in reactive oxygen intermediates (ROI) and 2) an elevation of Deltapsim. These events are followed by externalization of phosphatidylserine (PS), disruption of Deltapsim, and cell death. The caspase inhibitor peptides, DEVD-CHO, Z-VAD.fmk, and Boc-Asp.fmk, blocked Fas-induced PS externalization, disruption of Deltapsim, and cell death, suggesting that these events are sequelae of caspase activation. By contrast, in the presence of caspase inhibitors, ROI levels and Deltapsim of Fas-stimulated cells remained elevated. Because ROI levels and Deltapsim are regulated by the supply of reducing equivalents from the pentose phosphate pathway (PPP), we studied the impact of transaldolase (TAL), a key enzyme of the PPP, on Fas signaling. Overexpression of TAL accelerated Fas-induced mitochondrial ROI production, Deltapsim elevation, activation of caspase-8 and caspase-3, proteolysis of poly(A)DP-ribose polymerase, and PS externalization. Additionally, suppression of TAL diminished these activities. Therefore, by controlling the balance between mitochondrial ROI production and metabolic supply of reducing equivalents through the PPP, TAL regulates susceptibility to Fas-induced apoptosis. Early increases in ROI levels and Deltapsim as well as the dominant effect of TAL expression on activation of caspase-8/Fas-associated death domain-like IL-1beta-converting enzyme, the most upstream member of the caspase cascade, suggest a pivotal role for redox signaling at the initiation of Fas-mediated apoptosis.

OBJECTIVE

To study the effect of the systemic inflammatory response syndrome (SIRS) or major elective surgery on the apoptosis of circulating polymorphonuclear neutrophils because an activated inflammatory response is terminated, in part, through the programmed cell death, or apoptosis, of its effector cells.

METHODS

A prospective inception cohort study.

METHODS

A mixed surgical and medical intensive care unit of an adult tertiary care hospital.

METHODS

Sixteen patients with SIRS, 7 uninfected patients who had undergone elective aortic aneurysmectomy, and 8 healthy laboratory control subjects.

METHODS

Serial blood samples were drawn for evaluation of neutrophil apoptosis, activational state, and surface receptor expression by flow cytometry.

METHODS

Spontaneous apoptosis was significantly delayed in neutrophils from patients with SIRS (8.6%+/-6.8%) and patients who had undergone elective aortic aneurysmectomy (11.0%+/-5.0%) when compared with controls (34.9%+/-6.8%). These neutrophils were activated as evidenced by enhanced respiratory burst activity and augmented surface expression of CD11b. Apoptosis in response to engagement of cell surface Fas (also known as CD95 or APO-1) with an agonistic antibody was blunted. Plasma from patients with SIRS or patients who had undergone elective aortic aneurysmectomy suppressed the apoptotic responses of control neutrophils (plasma from patients with SIRS, 18.8%+/-10.3%; plasma from patients who had undergone elective aortic aneurysmectomy, 20.0%+/-6.1%; P<.01). Western blot analysis showed normal expression of the key proapoptotic proteases, interleukin 1beta converting enzyme and CPP32 (also known as YAMA, apopain, and caspase 3), indicating that delayed apoptosis was not a consequence of decreased levels of proapoptotic enzymes.

CONCLUSIONS

Circulating neutrophils from patients with SIRS or from patients who have undergone major elective surgery show delayed expression of constitutive programmed cell death, and antiapoptotic factors are present in the general circulation. While prolonged neutrophil survival may represent an appropriate adaptive response to injury, the presence of activated and apoptosis-resistant cells in an antiapoptotic environment may contribute to the systemic inflammatory injury characteristic of SIRS and predispose to the development of the multiple organ dysfunction syndrome.

Adipose tissue inflammation is linked to the pathogenesis of insulin resistance. In addition to exerting death-promoting effects, the death receptor Fas (also known as CD95) can activate inflammatory pathways in several cell lines and tissues, although little is known about the metabolic consequence of Fas activation in adipose tissue. We therefore sought to investigate the contribution of Fas in adipocytes to obesity-associated metabolic dysregulation. Fas expression was markedly increased in the adipocytes of common genetic and diet-induced mouse models of obesity and insulin resistance, as well as in the adipose tissue of obese and type 2 diabetic patients. Mice with Fas deficiency either in all cells or specifically in adipocytes (the latter are referred to herein as AFasKO mice) were protected from deterioration of glucose homeostasis induced by high-fat diet (HFD). Adipocytes in AFasKO mice were more insulin sensitive than those in wild-type mice, and mRNA levels of proinflammatory factors were reduced in white adipose tissue. Moreover, AFasKO mice were protected against hepatic steatosis and were more insulin sensitive, both at the whole-body level and in the liver. Thus, Fas in adipocytes contributes to adipose tissue inflammation, hepatic steatosis, and insulin resistance induced by obesity and may constitute a potential therapeutic target for the treatment of insulin resistance and type 2 diabetes.

Induction of apoptosis by oncogenes like c-myc may be important in restraining the emergence of neoplasia. However, the mechanism by which c-myc induces apoptosis is unknown. CD95 (also termed Fas or APO-1) is a cell surface transmembrane receptor of the tumor necrosis factor receptor family that activates an intrinsic apoptotic suicide program in cells upon binding either its ligand CD95L or antibody. c-myc-induced apoptosis was shown to require interaction on the cell surface between CD95 and its ligand. c-Myc acts downstream of the CD95 receptor by sensitizing cells to the CD95 death signal. Moreover, IGF-I signaling and Bcl-2 suppress c-myc-induced apoptosis by also acting downstream of CD95. These findings link two apoptotic pathways previously thought to be independent and establish the dependency of Myc on CD95 signaling for its killing activity.

Here we investigated the cytotoxicity of JS-K, a prodrug designed to release nitric oxide (NO(*)) following reaction with glutathione S-transferases, in multiple myeloma (MM). JS-K showed significant cytotoxicity in both conventional therapy-sensitive and -resistant MM cell lines, as well as patient-derived MM cells. JS-K induced apoptosis in MM cells, which was associated with PARP, caspase-8, and caspase-9 cleavage; increased Fas/CD95 expression; Mcl-1 cleavage; and Bcl-2 phosphorylation, as well as cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (EndoG) release. Moreover, JS-K overcame the survival advantages conferred by interleukin-6 (IL-6) and insulin-like growth factor 1 (IGF-1), or by adherence of MM cells to bone marrow stromal cells. Mechanistic studies revealed that JS-K-induced cytotoxicity was mediated via NO(*) in MM cells. Furthermore, JS-K induced DNA double-strand breaks (DSBs) and activated DNA damage responses, as evidenced by neutral comet assay, as well as H2AX, Chk2 and p53 phosphorylation. JS-K also activated c-Jun NH(2)-terminal kinase (JNK) in MM cells; conversely, inhibition of JNK markedly decreased JS-K-induced cytotoxicity. Importantly, bortezomib significantly enhanced JS-K-induced cytotoxicity. Finally, JS-K is well tolerated, inhibits tumor growth, and prolongs survival in a human MM xenograft mouse model. Taken together, these data provide the preclinical rationale for the clinical evaluation of JS-K to improve patient outcome in MM.