Role of apoptosis in HIV disease pathogenesis

After approximately one and a half decades of intensive studies, the exact mechanisms to explain HIV-mediated cytopathicity are still enigmatic and need closer scrutiny. There has been a dichotomy between virological and immunological viewpoints in understanding HIV-mediated cytopathicity, the former emphasizing a killing of infected cells by HIV-1 and the latter emphasizing indirect mechanisms wherein HIV or its soluble component(s) alter CD4 T-cell function and induce susceptibility to apoptosis. Accumulating evidence points to the notion that apoptosis might be a major contributor to the depletion of CD4 T-cells in HIV infection. This review summarizes current information about the regulatory mechanisms of T-cell apoptosis and the role of apoptosis in HIV pathogenesis with the goal of providing an integrated view of HIV cytopathicity.     

Comparison of the TUNEL,lamin B and annexin V methods for the detection of apoptosis by flow cytometry

The present study was performed in order to compare the sensitivity of 3 apoptosis-detection methods using a model cell culture after induction of apoptosis. Evaluation was performed by means of flow cytometry and light microscopy. It appeared that both TUNEL and annexin V methods are sensitive and specific and produced similar data in all measurements. The immunocytochemical detection of lamin B was less reliable than the other methods.     

Anti-retroviral therapy reverses HIV-associated abnormalities in lymphocyte apoptosis

The objective of this study was to assess the role of anti-retroviral therapy (ART) on the susceptibility of peripheral blood lymphocytes (PBL) from HIV-1-infected individuals to activation-induced apoptosis and in comparison with changes in CD4 lymphocyte counts. Eleven symptomatic HIV⁺ patients were studied. Ex vivo apoptosis was measured in phytohaemagglutinin (PHA)-stimulated PBL and CD4 subsets by flow cytometry, at baseline and after 1 month (4–6 weeks) and 2/3 months of ART. Six patients had extended studies of the effects of therapy to a maximum of 21 months. Lymphocyte apoptosis was significantly elevated in HIV⁺ patients at baseline (median 22% compared with 7.5% in HIV^? risk-matched controls; P < 0.05). This decreased to control levels on ART (7.4% at 4–6 weeks, P < 0.01, and 6.2% at 8–12 weeks, P < 0.05, compared with baseline). Similar changes occurred in the CD4⁺ subpopulation. The decrease in apoptosis was maintained for several months, but the effect was rapidly lost if ART was discontinued. CD4 counts showed a reciprocal relationship to changes in apoptosis. The association of changes in apoptosis with those in CD4 counts suggests a link between programmed cell death and lymphocyte depletion. Apoptosis reduced in some individuals without any reduction in viral load, suggesting apoptosis may be influenced by factors in addition to the overall extent of HIV replication.     

Increased Fas-mediated apoptosis in polymorphonuclear cells from HIV-infected patients

Neutrophils represent an important line of innate host defence against invading microorganisms and their functional detriment during HIV infection, including accelerated spontaneous cell death, has been shown to contribute to AIDS development. Neutrophils are susceptible to apoptosis via Fas and an interaction between Fas and FasL was suggested originally as a mechanism to explain constitutive neutrophil apoptosis. We have explored some intracellular pathways leading to PMN apoptosis from 28 HIV-infected patients and 24 healthy volunteers. As previously reported, accelerated spontaneous apoptosis was observed in HIV+ patients, but this did not correlate with viral load. Furthermore, an increase in the level of spontaneous apoptosis was detected in neutrophils from HIV-infected patients following inhibition of ERK, suggesting an impairment of this kinase pathway during the early stages of infection which may contribute to PMN dysfunction. An elevated susceptibility to undergo apoptosis was observed following cross-linking of Fas, which correlated both with viral load and co-expression of Fas/FasL surface molecules. Different mechanisms for spontaneous and Fas-induced apoptosis are proposed which together contribute to the neutropenia and secondary infections observed during the progression to AIDS.     

Thalidomide analogue CC-3052 reduces HIV+ neutrophil apoptosis in vitro

Thalidomide has significant immunomodulatory properties and has been used successfully in the treatment of oral ulcers and wasting in HIV patients. However, its use is limited by its poor bioavailability due to low solubility and short half life in solution, and teratogenic and neurotoxic side-effects. Recently, water-soluble analogues of thalidomide with significantly greater immunomodulatory activity and reduced side-effects have become available. We examined the effect of thalidomide and one analogue, CC-3052, on neutrophil apoptosis following culture for 20 h in vitro. Apoptosis was assessed by reduced CD16 expression and Annexin V binding using flow cytometry. Thalidomide or CC-3052 alone had no effect on neutrophil apoptosis when used at physiological levels. However, when used together with prostaglandin E2 (10-7 M), a potent adenylate cyclase activator, CC-3052 but not thalidomide (both 10-5 M) reduced apoptosis in neutrophils from normal and HIV+ donors. The reduced apoptosis could not be attributed to the ability of CC-3052 to reduce tumour necrosis factor-alpha (TNF-alpha) production, but may be due to its PDE4 inhibitor properties, as it increased [cAMP]i, and mimicked the effect of increasing [cAMP]i using dibutryl cAMP, a membrane-permeable analogue of cAMP. The results suggest a role for thalidomide analogue CC-3052 in reducing persistent activation of the TNF-alpha system in HIV without markedly impairing neutrophil viability.     

Caspase 3反义核酸阻抑Aβ25～35诱导的细胞凋亡的研究

目的 :设计、合成Caspase 3mRNA反义寡核苷酸 ,并筛选出能阻抑Aβ2 5～ 3 5诱导的PC 12细胞凋亡的有效片段。方法 :针对Caspase 3mRNA不同区域设计出一系列 17～ 18 mer反义寡核苷酸 ,用脂质体导入PC 12细胞内 ,并用Aβ2 5～ 3 5诱导细胞凋亡。用AnnexinⅤ FITC/PI复染、流式细胞术检测观察凋亡早期细胞变化情况。结果 :针对Caspase 3mRNA编码起始区的反义寡核苷酸片段 (ASODN2 ,-6～ 12 ,5’ GTTGTTGTCCATGGTCAC 3’)导入PC 12细胞后 ,在Aβ2 5～ 3 5诱导下 ,AnnexinⅤ /PI复染、流式细胞仪检测见早期凋亡细胞较对照组及其它反义寡核苷酸组明显减少 (P <0 .0 5 ) ;且导入后Aβ2 5～ 3 5诱导凋亡细胞数与无诱导组相比未见明显增多 (P >0 .0 5 )。结论 :1.Caspase 3在Aβ2 5～ 3 5诱导的PC 12细胞凋亡中起重要作用 ;2 .Caspase 3mRNA的反义寡核苷酸片段 5’ GTTGTTGTCCATGGTCAC 3’能有效阻止Aβ2 5～ 3 5诱导的PC 12细胞凋亡     

Costimulatory pathways in transplantation

Secondary, so-called costimulatory, signals are critically required for the process of T cell activation. Since landmark studies defined that T cells receiving a T cell receptor signal without a costimulatory signal, are tolerized in vitro, the investigation of T cell costimulation has attracted intense interest. Early studies demonstrated that interrupting T cell costimulation allows attenuation of the alloresponse, which is particularly difficult to modulate due to the clone size of alloreactive T cells. The understanding of costimulation has since evolved substantially and now encompasses not only positive signals involved in T cell activation but also negative signals inhibiting T cell activation and promoting T cell tolerance. Costimulation blockade has been used effectively for the induction of tolerance in rodent models of transplantation, but turned out to be less potent in large animals and humans. In this overview we will discuss the evolution of the concept of T cell costimulation, the potential of ‘classical’ and newly identified costimulation pathways as therapeutic targets for organ transplantation as well as progress towards clinical application of the first costimulation blocking compound.     

Effects of therapy with highly active anti-retroviral therapy (HAART) and IL-2 on CD4+ and CD8+ lymphocyte apoptosis in HIV+ patients

The kinetics and effects of in vivo spontaneous apoptosis and activation-induced cell death (AICD) upon CD4+ and CD8+ lymphocyte subsets and CD4 naive cell numbers were studied in HIV+ subjects with CD4 pretreatment values > 200/mm3, who were subsequently treated for 48 weeks with HAART alone or in combination with six cycles of subcutaneous IL-2. Irrespective of the type of treatment, patients showed a statistically significant increase in CD4 cell counts after 4 weeks, although the CD4 naive subset only increased significantly in the IL-2-treated subjects at the end of treatment. The percentage of CD4 cells undergoing spontaneous apoptosis and AICD was significantly reduced in all patients after 4 weeks and this reduction was maintained until the end of therapy; however, the level always remained significantly higher in comparison with healthy subjects. A statistically significant reduction in CD8 apoptosis levels required at least 24 weeks of therapy. Together these data suggest that a reduction in the level of apoptosis may contribute to the early rise in CD4 numbers measured after HAART, but that later on HAART is unable to improve further this biological parameter. Although the use of IL-2 had no additional effects on spontaneous apoptosis and AICD, it may be beneficial by stimulating a late increase in the numbers of CD4 naive cells in HIV-treated subjects.     

Fas与体外胸腺细胞自发凋亡的关系

目的 探讨体外培养的胸腺细胞表达Fas、Fas-L与自发凋亡的关系。方法 应用流式细胞术,检测体外培养不同时间的胸腺细胞的凋亡率及Fas、Fas-L表达的阳性率。结果 体外培养的胸腺细胞自发凋亡率和Fas表达阳性率,均呈时间依赖性增加,Fas-L在体外培养24h时明显增加,体外培养的胸腺细胞Fas的表达与自发凋亡明显相关。结论 Fas是介导体外培养的胸腺细胞自发凋亡的重要分子。     

Activation-induced peripheral blood T cell apoptosis is Fas independent in HIV-infected individuals

T cell apoptosis has been proposed as an Important contributorto the functional defects and depletion of T cells in HIV-lnfectedIndividuals. However, the mechanisms Involved in this apoptosishave not been elucidated. We recently showed that peripheralblood T cells from HIV-infected individuals are especially susceptibleto Fas antigen-induced apoptosis. In this study we examine therole of Fas, CTLA-4, tumor necrosis factor (TNF) receptors (TNFR)and CD30, receptors known to be involved In T cell activation-inducedcell death (AICD), in the spontaneous and activation (anti-CD3)-lnducedapoptosis of peripheral blood T cells from asymptomatic HIV-infectedindividuals. We report here that spontaneous and activation-InducedT cell apoptosis cannot be inhibited by reagents that blockinteractions of Fas, CTLA-4, p55 and p75 TNFR and CD30 withtheir respective ligands. We also show that IL-12, IFN-, IL-4and IL-10 cannot modify spontaneous, activation- and anti-Fas-inducedapoptosis. Anti-Fas preferentially Induced CD4⁺ T cell apoptosiswhereas AICD induced apoptosis equally In CD4⁺ and CD8⁺ T cells.We conclude that T cell AICD In HIV infection Is not mediatedby Fas, thus Indicating that Fas-Induced and activation-InducedT cell apoptosis are independent mechanisms of apoptosis whichmay play different roles in the pathogenesls of HIV infection.     

Colchicine-induced apoptosis and anti-Fas localization in rat-incisor ameloblasts

Apoptosis of ameloblasts were examined by the TdT-mediated dUTP-biotin nick end-labelling method and electron microscopy 8 h after injection of colchicine. The results showed that extensive apoptosis occurred in ameloblasts of secretion to maturation zones. To determine the possible involvement of stimulators in ameloblast apoptosis, Fas, Fas ligand, tumor-necrosis-factor α, and tumor-necrosis-factor receptor 1 were examined utilizing immunohistochemistry and Western blotting analysis. Only Fas was consistently detected in the secretion, transition and maturation ameloblasts and overlying enamel organ epithelia. These results suggest that ameloblasts could undergo apoptosis by colchicine and that one of the ameloblast apoptosis mediators would be the Fas receptor.     

Low-dose IL-2 reduces lymphocyte apoptosis and increases naive CD4 cells in HIV-1 patients treated with HAART

During HIV disease an increased in vitro apoptosis of peripheral blood mononuclear cells has been demonstrated. This can be reversed in vitro by interleukin (IL)-2. Recent trials with highly active antiretroviral therapy (HAART) and IL-2 in HIV-1-infected patients showed promising immunological and clinical results. Here we investigated the effects of subcutaneous low-dose IL-2 administration in combination with HAART on in vitro apoptosis and the relationship between apoptosis, CD4(+) counts, and HIV replication. Twenty-two asymptomatic HIV patients were randomized for HAART (arm I) and HAART plus IL-2 (arm II). Spontaneous apoptosis was decreased in both arms after 28 weeks of therapy but the reduction was highly significant only in arm II (P = 0.05 vs P = 0.001). As the percentage of apoptosis decreased, there was a significantly higher increase of both CD4(+) and CD4(+) naive T cells in arm II vs arm I. HIV plasma viremia was reduced in all patients after therapy. Our data suggest that intermittent therapy with low-dose subcutaneous IL-2 in addition to HAART induces a positive immunomodulation in asymptomatic HIV-infected patients.     

Participation of the Fas and Fas ligand systems in apoptosis during atrophy of the rat submandibular glands

Most acinar cells and some duct cells undergo apoptosis during atrophy of the submandibular gland. The present study was designed to elucidate whether Fas and its receptor ligand (FasL) are involved during apoptotic atrophy of the gland. The excretory duct of the right submandibular gland of rats was doubly ligated with metal clips from 1 to 14 days for induction of gland atrophy. Control rats were untreated. Fas and FasL expression in the atrophied submandibular gland was detected using immunohistochemistry (IHC) and Western immunoblot. Expression of activated caspase 8 and activated caspase 3 was also detected with IHC. Fas-positive acinar and duct cells and FasL-positive duct cells increased in the atrophic glands at 3 and 5 days after duct ligation when apoptotic cells were commonly observed. Thereafter, Fas- and FasL-positive cells declined in number. Patterns of expression of Fas and FasL using Western immunoblots concurred with the IHC results. Activated caspase 8-positive cells were present at every time interval but peaked at 3 and 5 days following duct ligation. The cells showing immunoreaction for activated caspase 3 first appeared on day 3, with the peak in apoptosis, after which they decreased. The results indicate that the Fas/FasL systems likely play an important role in apoptotic pathways during atrophy of the submandibular gland.     

Fas-FasL和caspase-3信号通路在启动SLE患者T细胞亚群凋亡中的作用

目的：探讨SLE患者T细胞亚群的Fas-FasL信号传导通路与T细胞凋亡紊乱的关系。方法：应用流式细胞术测定T细胞亚群表面Fas、FasL的表达率及细胞质中活化caspase-3的表达率。结果：与健康对照组相比较，活动期及稳定期SLE患者组CD4^＋T细胞表面Fas的表达率均显著增加（P〈0．05），CD8^＋T细胞表面Fas的表达率略有增加但无统计学意义（P〉0．05）。稳定期和活动期SLE患者组T细胞亚群表面FasL的表达率均显著增加（P〈0．05），但两疾病组问T细胞亚群表面Fas、FasL的表达率无显著性差异（P〉0．05）。活动期SLE患者组T细胞亚群细胞质中活化caspase-3的表达率，明显高于稳定期SLE患者组和健康对照组（P〈0．05）。稳定期SLE患者组T细胞亚群细胞质中活化caspase-3的表达率略高于健康对照组，但无统计学意义。结论：SLE患者外周血T细胞亚群凋亡加速，CC4^＋ T细胞的凋亡活跃，其中Fas-FasL信号传导途径可能起重要的作用。T细胞凋亡紊乱的程度与SLE的活动程度密切相关。     

Enhancement of ectopic bone formation in mice with a deficit in Fas-mediated apoptosis

Bone tormation is under the control of cytokines as well as growth factors such as bone morphogenetlc protelns (BMP). This suggests the possibillity that osteogenesls might be modulated by factors which atso modulate the Immune system. To test whether Immune disorders In the host may influence bone formation, we studied BMP-Induced bone formation In a C3H/HeJ strain of mice benring a mutant gene, the lymphoproliteration Qene ( lpr ) or the genemlbed lym-phoprolifarative diseaee gene (gld), both of which are known to be a Fas delaion mutant and a Fas ligand mutant, respectively, and to Induce Immune disorders vla a deficit In Fas-mediated apoptoak Crude BMP derived from bovine bone were injscted into the muscular tlasue In the femur of adult C3H/HaJ mice or C3H/HeJ mice bearing an lpr or gld gene. Quantltathre analysis of the resulting ectopic bone formation by X-ray photography 2 weeks after infection revealed that the presence of either the Ipr or gld gene caused a bone mess dgnlficantly larger In dimension than that seen in the wiid type mice. Histologlcal examlnatlon also revealed the dmerent Influence between these mutant genes on the level of bone fofmatlon exhibited by hyallne cartilage and bone imbeculae. Based on these results, we discussed the possible mechanisms of the enhanced ectopic bone fotmation under the deficit In Fas-medlated apoptosls.     

Hydroxychloroquine potentiates Fas-mediated apoptosis of rheumatoid synoviocytes

Inadequate apoptosis may contribute to the synovial hyperplasia associated with rheumatoid arthritis (RA). The Fas-associated death domain protein (FADD)-like interleukin (IL)-1beta-converting enzyme (FLICE)-inhibitory protein (FLIP), which is an apoptotic inhibitor, has been implicated in the resistance to Fas-mediated apoptosis of synoviocytes. This study investigated whether hydroxychloroquine (HCQ), an anti-rheumatic drug, induces the apoptosis of rheumatoid synoviocytes, and modulates the expression of FLIP. Fibroblast-like synoviocytes (FLS) were prepared from the synovial tissues of RA patients, and were cultured with various concentrations of HCQ in the presence or absence of the IgM anti-Fas monoclonal antibodies (mAb) (CH11). Treatment with HCQ, ranging from 1 to 100 microM, induced the apoptosis of FLS in a dose- and time-dependent manner. The increase in synoviocytes apoptosis by HCQ was associated with caspase-3 activation. A combined treatment of HCQ and anti-Fas mAb increased FLS apoptosis and caspase-3 activity synergistically, compared with either anti-Fas mAb or HCQ alone. The Fas expression level in the FLS was not increased by the HCQ treatment, while the FLIP mRNA and protein levels were decreased rapidly by the HCQ treatment. Moreover, time kinetics analysis revealed that the decreased expression of FLIP by HCQ preceded the apoptotic event that was triggered by HCQ plus anti-Fas mAb. Taken together, HCQ increases the apoptosis of rheumatoid synoviocytes by activating caspase-3, and also sensitizes rheumatoid synoviocytes to Fas-mediated apoptosis. Our data suggest that HCQ may exert its anti-rheumatic effect in rheumatoid joints through these mechanisms.     

Increased expression of FLIP,an inhibitor of Fas-mediated apoptosis,in stomach cancer

Despite the cell surface expression of Fas (Apo-1/CD95), many types of tumor cells, including stomach cancer cells, are resistant to Fas-mediated apoptosis, indicating the presence of inactivating mechanisms of Fas signaling. Expression of FLICE-like inhibitory protein (FLIP), one of the inhibitory proteins of Fas-mediated apoptosis, has been reported in several cancer types, but not in stomach cancer. In the present study, we analyzed the expression of Fas and FLIP in 60 advanced gastric adenocarcinomas by immunohistochemistry using a tissue microarray approach. Immunopositivity (defined as >/=30% of the neoplastic cells) was observed for Fas in 58 (97%) and FLIP in 54 (90%) of the 60 cancers. All of the tumors with FLIP immunostaining also showed Fas immunostaining. Loss of cell surface Fas immunostaining, another mechanism of Fas resistance, was observed in 45 tumors (75%). By contrast, normal gastric mucosal cells showed no or weak expression of both Fas and FLIP. Taken together, these results indicate that increased expression of FLIP is a frequent event in stomach carcinomas, and suggest that for evading apoptosis stomach carcinoma cells in vivo may need FLIP expression, which might contribute to tumor development.     

Staphylococcus aureus, but not Staphylococcus epidermidis, modulates the oxidative response and induces apoptosis in human neutrophils

S. epidermidis is the most common isolate in foreign body infections. The aim of this study was to understand why S. epidermidis causes silent biomaterial infections. In view of the divergent inflammatory responses S. epidermidis and S. aureus cause in patients, we analyzed how they differ when interacting with human neutrophils. Neutrophils interacting with S. epidermidis strains isolated either from granulation tissue covering infected hip prostheses or from normal skin flora were tested by measuring the oxidative response as chemiluminescence and apoptosis as annexin V binding. Different S. aureus strains were tested in parallel. All S. epidermidis tested were unable to modulate the oxidative reaction in response to formyl-methionyl-leucyl-phenylalanine (fMLP) and did not provoke, but rather inhibited, apoptosis. In contrast, some S. aureus strains enhanced the oxidative reaction, and this priming capacity was linked to p38-mitogen-activated-protein-kinase (p38-MAPK) activation and induction of apoptosis. Our results may explain why S. epidermidis is a weak inducer of inflammation compared to S. aureus, and therefore responsible for the indolent and chronic course of S. epidermidis biomaterial infections.