Endothelial damage in all types of T-lymphocyte-mediated drug-induced eruptions.

BACKGROUND: In severe drug-induced eruptions, bullous lesions can be associated with immune complex-mediated vasculitis and/or with T-lymphocyte-mediated keratinocyte apoptosis. We have recently identified endothelial cell apoptosis in severe bullous T-lymphocyte-mediated drug-induced eruptions. We assessed microvessel involvement in the whole spectrum of T-lymphocyte-mediated drug-induced eruptions. Thirty-two patients with T-lymphocyte-mediated drug-induced eruptions in 4 groups (8 cases of toxic epidermal necrolysis/Stevens-Johnson syndrome, 8 cases of drug rash with eosinophilia and systemic symptoms, 8 cases of acute generalized exanthematous pustulosis, 8 cases of drug maculopapular exanthema) and 8 healthy controls were included. On skin biopsy specimens, we performed a systematic ultrastructural study of endothelial cells, vascular walls, and inflammatory cells; a quantification of apoptotic cells, inflammatory infiltrate, and immune complex deposits; and we assessed granzyme-B, tumor necrosis factor, and Fas ligand expression. Correlations of apoptosis with clinical data of skin lesions and systemic involvement in liver, kidney, lung, and lymph nodes were then assessed. OBSERVATIONS: Findings from ultrastructural study showed that endothelial cell apoptosis was present in all 32 drug-induced eruptions. No leukocytoclastic vasculitis was associated. Granzyme-B and tumor necrosis factor were expressed around microvessels. In toxic epidermal necrolysis/Stevens-Johnson syndrome and drug rash with eosinophilia and systemic symptoms, the number of apoptotic endothelial cells was related to the extension of skin lesions and the presence of purpura. It was also related to liver and kidney involvement. CONCLUSIONS: Endothelial apoptosis occurs in skin microvessels of all types of T-lymphocyte-mediated drug-induced eruptions. This skin endothelial cell damage is related to the severity of skin lesions and systemic involvement.

Bcl-xL gene expression correlated with lower apoptotic cell numbers and shorter progression-free survival in PCFCL.

The expression of bcl-x(L), an antiapoptotic member of the bcl-2 family, has been correlated with poor prognosis in nodal follicular lymphomas (NFLs). So far, it has not been studied in primary cutaneous follicle center lymphomas (PCFCLs), which, compared with NFLs, express less frequently t(14;18)(q32;q21) and bcl-2. Using real-time PCR we measured bcl-xL and bcl-2 gene expression levels in laser-microdissected lymphoma cells of 20 PCFCL frozen sections. Numbers of apoptotic cells labeled by TUNEL assay were negatively correlated with bcl-xL expression levels (r=-0.840, P<0.005). Bcl-xL expression was significantly higher in biopsies of patients who developed relapse or disease progression later compared with patients who did not (P=0.022), and higher levels of bcl-xL gene expression were significantly correlated with shorter progression-free survival (PFS) (P=0.017). None of these features was correlated with bcl-2 gene expression levels. Our findings indicate that bcl-xL overexpression is inversely correlated with PFS in PCFCL. Moreover, the inverse correlation between bcl-xL expression levels and apoptotic cell numbers suggests that bcl-xL, through its antiapoptotic effect, might contribute to tumor cell survival in PCFCL.

The human cytomegalovirus-encoded chemokine receptor US28 induces caspase-dependent apoptosis.

Viral subversion of apoptosis regulation plays an important role in the outcome of host/virus interactions. Although human cytomegalovirus (HCMV) encodes several immediate early (IE) antiapoptotic proteins (IE1, IE2, vMIA and vICA), no proapoptotic HCMV protein has yet been identified. Here we show that US28, a functional IE HCMV-encoded chemokine receptor, which may be involved in both viral dissemination and immune evasion, constitutively induces apoptosis in several cell types. In contrast, none of nine human cellular chemokine receptors, belonging to three different subfamilies, induced any significant level of apoptosis. US28-induced cell death involves caspase 10 and caspase 8 activation, but does not depend on the engagement of cell-surface death receptors of the tumour necrosis factor receptor/CD95 family. US28 cell-death induction is prevented by coexpression of C-FLIP, a protein that inhibits Fas-associated death domain protein (FADD)-mediated activation of caspase 10 and caspase 8, and by coexpression of the HCMV antiapoptotic protein IE1. The use of US28 mutants indicated that the DRY sequence of its third transmenbrane domain, required for constitutive G-protein signalling, and the US28 intracellular terminal domain required for constitutive US28 endocytosis, are each partially required for cell-death induction. Thus, in HCMV-infected cells, US28 may function either as a chemokine receptor, a phospholipase C activator, or a proapoptotic factor, depending on expression levels of HCMV and/or cellular antiapoptotic proteins.

Mechanisms involved in the low-level regeneration of CD4+ cells in HIV-1-infected patients receiving highly active antiretroviral therapy who have prolonged undetectable plasma viral loads.

BACKGROUND: Persistent low CD4(+) cell counts are observed in 5%-27% of patients treated for human immunodeficiency virus (HIV)-1 infection despite their having prolonged undetectable plasma viral loads. METHODS: To understand the possible mechanisms of this discordant immunological situation, a prospective transsectional case-control study was designed. HIV-1-infected subjects who had a plasma viral load <200 copies/mL for >1 year were considered to be case patients if their CD4(+) cell count was <250/mm(3); control patients had CD4(+) cell counts >500/mm(3) and were matched by sex, age, and nadir CD4(+) cell count to case patients. T cell proliferation after stimulation with various antigens, T cell subset counts, T cell rearrangement excision circles (TRECs), T cells undergoing apoptosis, cytokines influencing apoptosis, and cellular proviral DNA and plasma viral RNA persistence were assessed. RESULTS: Compared with the 19 control patients, the 19 case patients had undistinguishable lymphoproliferative responses to candidin and cytomegalovirus, fewer naive CD4(+) cells (CD45RA(+)62L(+), 23%+/-13% vs. 47%+/-14%; P<.0001), lower thymic output (1.28 vs. 3.95 TRECs/microL of blood; P=.0015), increased cell death by apoptosis (spontaneous, 23.2%+/-8.3% vs. 11.9%+/-8.4% [P=.02]; Fas induced, 38.6%+/-13.7% vs. 16.4%+/-8.0% [P=.004]), higher levels of plasma soluble tumor necrosis factor receptor II (9.6 vs. 5.3 ng/mL; P=.0058), and undistinguishable plasma HIV-1 and cellular proviral DNA loads. CONCLUSIONS: The mechanisms responsible for the low-level regeneration of CD4(+) cells involve, at least, deficiency in the regeneration of central CD4(+) cells and excessive apoptosis.

Interleukin 7 increases human immunodeficiency virus type 1 LAI-mediated Fas-induced T-cell death.

Fas-mediated T-cell death is known to occur during human immunodeficiency virus (HIV) infection. In this study, we found that HIV type 1 LAI (HIV-1(LAI)) primes CD8(+) T cells from healthy donors for apoptosis, which occurs after Fas ligation. This effect is counteracted by a broad caspase inhibitor (zVAD-fmk). Fas-mediated cell death does not depend on CD8(+) T-cell infection, because it occurred in the presence of reverse transcriptase inhibitors. However, purified CD8(+) T cells are sensitive to Fas only in the presence of soluble CD4. Finally, we found that interleukin 7 (IL-7) increases Fas-mediated CD4(+) and CD8(+) T-cell death induced by HIV-1(LAI). Since high levels of IL-7 are a marker of poor prognosis during HIV infection, our data suggest that enhancement of Fas-mediated T-cell death by HIV-1(LAI) and IL-7 is one of the mechanisms involved in progression to AIDS.

On the evolution of erythrocyte programmed cell death: apoptosis of Rana esculenta nucleated red blood cells involves cysteine proteinase activation and mitochondrion permeabilization.

Batracian Rana esculenta erythrocytes cell death induced by either calcium influx, or staurosporine, involves typical apoptotic phenotype. Our data reveal: (i) a drastic modification of the cell morphology with loss of the ellipsoidal form as assessed by phase contrast microscopy and scanning electron microscopy; (ii) an exposure of the phosphatidylserine residues in the outer leaflet of the cell membrane; (iii) a caspase-3-like activity; (iv) a mitochondrial membrane potential (Delta Psi m) loss; and (v) a chromatin condensation and fragmentation. Erythrocyte chromatin condensation and fragmentation are prevented by caspase and calpain peptide inhibitors. These inhibitors also prevent Delta Psi m loss supporting the idea that mitochondria is a central sensor for Rana erythrocytes cell death. Our observations highlight the conservation of the programmed cell death machinery in erythrocytes across kingdom.

Leishmania major-mediated prevention of programmed cell death induction in infected macrophages is associated with the repression of mitochondrial release of cytochrome c.

Leishmania are obligate, intracellular parasites of macrophages in their vertebrate hosts, including humans, in which they cause disease. Here, we report that in vitro infection with Leishmania major protects murine bone marrow-derived macrophages against programmed cell death (PCD) induced by deprival of macrophage-colony stimulating factor and delays PCD caused by treatment with staurosporine, a broad inducer of PCD. This preventive effect was observed in macrophages from L. major-susceptible BALB/c and L. major-resistant C57BL/6 mice, indicating that repression of PCD did not depend on genetic background-specific regulation of T helper cell type 1 (Th1)/Th2 cytokine secretion. Prevention of effector caspase activation and PCD was associated with a repression of mitochondrial release of cytochrome c and did not involve the nuclear factor-kappaB pathway. The capacity of L. major to delay PCD induction in the infected macrophages may have implications for Leishmania pathogenesis by favoring the invasion of its host and the persistence of the parasite in the infected cells.

Prognostic significance of bcl-xL gene expression and apoptotic cell counts in follicular lymphoma.

bcl-xL, a member of the Bcl-2 family, exerts an antiapoptotic effect on lymphocytes. To assess its clinical significance in patients with follicular lymphoma, realtime quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of bcl-xL gene expression was investigated in whole lymph node sections and laser-microdissected lymphoma cells of 27 patients. Compared with 10 patients with reactive follicular hyperplasia, the bcl-xL gene was overexpressed in patients with follicular lymphoma at a higher level in microdissected lymphoma cells. The bcl-xL gene level correlated with the number of apoptotic lymphoma cells labeled by terminal deoxytransferase-catalyzed DNA nick-end labeling (TUNEL) assays (r = -0.7736). Clinically, a high bcl-xL level was significantly associated with multiple sites of extranodal involvement (P =.0020), elevated lactate dehydrogenase level (P =.0478), and an International Prognostic Index indicating high risk (P =.0235). Moreover, bcl-xL gene overexpression was linked to short overall survival times (P =.0129). The value of bcl-xL gene expression as a prognostic marker in follicular lymphoma should thus be considered.

Prognostic value of apoptotic cells and infiltrating neutrophils in graft-versus-host disease of the gastrointestinal tract in humans: TNF and Fas expression.

The gastrointestinal (GI) tract is a major target in graft-versus-host disease (GvHD). In rodents both tumor necrosis factor (TNF) and Fas-dependent apoptosis have been shown to play a major role in GvHD lesions, but data in humans on TNF and Fas in situ expression are scarce. More recently, the role of non-T cells as GvHD effectors has also been suggested in experimental models. Here we report a detailed quantitative pathologic analysis in 95 patients who underwent gastroduodenal biopsy. This analysis included characterization and quantification of the cellular infiltrate, TNF, TNF receptors, and Fas in situ expression analyses and quantification of apoptotic cell numbers. TNF was expressed in all biopsies and it was highly specific for acute GvHD. In multivariate analysis, including pathologic factors only, increased early transplant-related mortality (TRM) was associated with the presence of more than 20 neutrophils per field. Factors affecting early and late TRM were then assessed by multivariate analyses including both pathologic and clinical factors. Increased day-90 TRM was associated with the presence of more than 5 apoptotic bodies per field within the cellular infiltrate, and with stage II or higher acute liver GvHD. One-year TRM associated with the same 2 factors and with chronic GvHD.

Mitochondrial release of apoptosis-inducing factor occurs downstream of cytochrome c release in response to several proapoptotic stimuli.

Mitochondrial outer membrane permeabilization by proapoptotic Bcl-2 family proteins, such as Bax, plays a crucial role in apoptosis induction. However, whether this only causes the intracytosolic release of inducers of caspase-dependent death, such as cytochrome c, or also of caspase-independent death, such as apoptosis-inducing factor (AIF) remains unknown. Here, we show that on isolated mitochondria, Bax causes the release of cytochrome c, but not of AIF, and the association of AIF with the mitochondrial inner membrane provides a simple explanation for its lack of release upon Bax-mediated outer membrane permeabilization. In cells overexpressing Bax or treated either with the Bax- or Bak-dependent proapoptotic drugs staurosporine or actinomycin D, or with hydrogen peroxide, caspase inhibitors did not affect the intracytosolic translocation of cytochrome c, but prevented that of AIF. These results provide a paradigm for mitochondria-dependent death pathways in which AIF cannot substitute for caspase executioners because its intracytosolic release occurs downstream of that of cytochrome c.

A suppressive effect of the adenovirus 5 protein E1B 55K on apoptosis induced by IL-3 deprivation and gamma-irradiation.

The murine IL-3-dependent myeloid cell line 32D undergoes a rapid death when deprived of interleukin-3 (IL-3), a process that is suppressed or delayed by the constitutive expression of Bcl-2 or the Bcl-2-related Bcl-xL survival protein. The adenovirus type 5 E1B region encodes an E1B 55K protein, that has been reported to bind and inactivate the p53 protein that plays an important role in the induction of apoptosis. In order to explore the potential effect of the E1B 55K protein on IL-3 deprival-induced cell death, we have established 32D cell lines overexpressing the adenovirus E1B 55K protein and compared its ability to modulate the cell death with that of the human Bcl-2 protein. We observed that E1B 55K, as Bcl-2, delays the cell death caused by either IL-3-deprivation or DNA damage induced by gamma-irradiation. Cell-cycle analysis after IL-3 deprivation indicated that surviving Bcl-2 transfectants accumulate predominantly in the G0/G1 phase of the cell cycle, while E1B 55K transfectants survive in both G0/G1 and the S and G2/M phases of the cell cycle. zVAD-fmk, a broad caspase inhibitor, prevented chromatin condensation and fragmentation, but not cell death, suggesting that IL-3 deprivation induces a cell death program in which the caspases are dispensable. In contrast, both E1B 55K and Bcl-2 allowed cell survival and prevented the typical features of programmed cell death, such as phosphatidyl-serine exposure, loss of mitochondrial membrane potential, and chromatin condensation and fragmentation. Our findings indicate that the adenovirus 5 E1B 55K protein has the capability to act as a survival factor, and suggest that E1B 55K exerts its effect upstream of the activation of effector caspases, by preventing the loss of mitochondrial membrane potential induced by IL-3 deprivation.

Effects of antiretroviral drugs on human immunodeficiency virus type 1-induced CD4(+) T-cell death.

Apoptosis of peripheral blood T cells plays an important role in the pathogenesis of human immunodeficiency virus (HIV) infection. In this study, we found that HIV type 1 (HIV-1) primes CD4(+) T cells from healthy donors for apoptosis, which occurs after CD95 ligation or CD3-T-cell receptor (TCR) stimulation. CD95-mediated death did not depend on CD4 T-cell infection, since it occurred in the presence of the reverse transcriptase inhibitor didanosine (ddI). In contrast, apoptosis induced by productive infection (CD3-TCR stimulation) is prevented by both CD95 decoy receptor and ddI. Our data suggest that HIV-1 triggers at least two distinct death pathways: a CD95-dependent pathway that does not require viral replication and a viral replication-mediated cell death independent of the CD95 pathway. Further experiments indicated that saquinavir, a protease inhibitor, at a 0.2 microM concentration, decreased HIV-mediated CD95 expression and thus cell death, which is independent of its role in inhibiting viral replication. However, treatment of peripheral blood mononuclear cells from healthy donors with a higher concentration (10 microM) of an HIV protease inhibitor, saquinavir or indinavir, induced both a loss in mitochondrial membrane potential (DeltaPsim) and cell death. Thus, protease inhibitors have the potential for both beneficial and detrimental effects on CD4(+) T cells independent of their antiretroviral effects.

CD95 engagement induces disseminated endothelial cell apoptosis in vivo: immunopathologic implications.

Fas (CD95) is a death receptor involved in apoptosis induction on engagement by Fas ligand (CD95L). Although CD95L-mediated apoptosis has been proposed as a pathogenic mechanism in a wide range of diseases, including graft-versus-host disease, systemic CD95 engagement in mice by agonistic CD95-specific antibodies or by soluble multimeric CD95L (smCD95L), though lethal, has been reported to cause apoptosis only in a limited range of cell types, that is, hepatocytes, hepatic sinusoidal endothelial cells, and lymphocytes. Another member of the tumor necrosis factor (TNF)/CD95L family, TNF-alpha, induces disseminated vascular endothelial cell apoptosis, which precedes apoptosis of other cell types and lethal multiorgan failure. Here we show that systemic CD95 engagement in vivo by agonistic CD95-specific antibody or smCD95L causes rapid, extensive, and disseminated endothelial cell apoptosis throughout the body, by a mechanism that does not depend on TNF-alpha. Disseminated endothelial cell apoptosis was also the first detectable lesion in a murine model of acute tissue damage induced by systemic transfer of allogeneic lymphocytes and did not occur when allogeneic lymphocytes were from CD95L-defective mice. Both vascular and additional tissue lesions induced by agonistic CD95-specific antibody, smCD95L, or allogeneic lymphocytes were prevented by treatment with an inhibitor of caspase-8, the upstream caspase coupled to CD95 death signaling. Vascular lesions are likely to play an important role in the pathogenesis of allogeneic immune responses and of other diseases involving circulating CD95L-expressing cells or smCD95L, and the prevention of CD95-mediated death signaling in endothelial cells may have therapeutic implications in these diseases.

Productive HIV-1 infection of primary CD4+ T cells induces mitochondrial membrane permeabilization leading to a caspase-independent cell death.

We have explored in vitro the mechanism by which human immunodeficiency virus, type 1 (HIV-1) induces cell death of primary CD4+ T cells in conditions of productive infection. Although HIV-1 infection primed phytohemagglutinin-activated CD4+ T cells for death induced by anti-CD95 antibody, T cell death was not prevented by a CD95-Fc decoy receptor, nor by decoy receptors of other members of the TNFR family (TNFR1/R2, TRAILR1/R2/OPG, TRAMP) or by various blocking antibodies, suggesting that triggering of death receptors by their cognate ligands is not involved in HIV-induced CD4 T cell death. HIV-1 induced CD4 T cell shrinkage, cell surface exposure of phosphatidylserine, loss of mitochondrial membrane potential (Deltapsim), and mitochondrial release of cytochrome c and apoptosis-inducing factor. A typical apoptotic phenotype (nuclear chromatin condensation and fragmentation) only occurred in around half of the dying cells. Treatment with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a broad spectrum caspase inhibitor, prevented nuclear chromatin condensation and fragmentation in HIV-infected CD4+ T cells and in a cell-free system (in which nuclei were incubated with cytoplasmic extracts from the HIV-infected CD4+ T cells). Nevertheless, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone did not prevent mitochondrial membrane potential loss and cell death, suggesting that caspases are dispensable for HIV-mediated cell death. Our findings suggest a major role of the mitochondria in the process of CD4 T cell death induced by HIV, in which targeting of Bax to the mitochondria may be involved.