Inhibition Of Lymphocyte Apoptosis Research Articles

Interleukin-6 Rescues Lymphocyte from Apoptosis and Exhaustion Induced by Chronic Hepatitis C Virus Infection.

Chronic hepatitis C virus (HCV) infection appears to trigger the onset of immune exhaustion and apoptosis to potentially assist viral persistence inside the host, eventually leading to exacerbated conditions of inflammation and hepatocarcinogenesis. Growing evidence suggests that spontaneous apoptosis of peripheral blood mononuclear cells (PBMCs) could be one of the potential immune impairment mechanisms in chronic viral infection. Interleukin-6 (IL-6) is a pleiotropic cytokine that plays an essential role in regulating immune and inflammatory responses. Owing to its known role in priming T cell growth, differentiation, and inhibition of lymphocyte apoptosis, we investigated the protective effect of IL-6 in rescuing lymphocytes from apoptosis and functional exhaustion in chronic HCV infection. The expression pattern of antiapoptotic (Mcl-1 and Bcl-2), proapoptotic (caspase-3 and Bim) genes along with interferon gamma (IFN-γ) and T cell inhibitory receptor (TIM-3) was analyzed before and after in vitro IL-6 treatment of patient-derived PBMCs. It was observed that the expression of antiapoptotic genes, Mcl-1 and Bcl-2 increased (threefolds and twofolds, respectively) and there was a considerable downregulation in T cell inhibitory receptor (TIM-3) and caspase-3. Similarly, the capacity of PBMCs to produce IFN-γ was also significantly increased (p < 0.001) depicting the promising nature of IL-6 in enhancing lymphocyte effector function. Summing it up, the study supports the positive role of IL-6 in rescuing PBMC population; however, the cytokine alone is not sufficient to sustain the adaptive immunity. It could be used as a potential candidate for combinational therapy along with other regulatory factors for ex vivo enhancement of lymphocyte and may help in moving one step toward adoptive T cell therapy in chronic HCV infection.

Immune alterations in lung cancer - the new therapeutic approach

Lung cancer is the main cause of cancer death worldwide. An advanced stage of the disease at the time of diagnosis, observed in the majority of cases, does not allow for introduction of radical treatment or makes the treatment ineffective. Lung cancer as a solid tumour with a very low antigenicity escapes immune surveillance, and cytotoxic cells attack. Cytotoxic lymphocytes play a key role in anticancer defence, but the population of these cells individually differs. Many suppressor and regulatory mechanisms inhibit the recognition of tumour antigens by dendritic and cytotoxic cell activation. The population of regulatory T cells (T regs) plays a crucial role in this inhibition of immune response. Their function depends on the expression of transcription factor Foxp3 and the presence of CTLA-4 molecules. The increased proportion of T regs and high expression of Foxp3 and CTLA-4 on lung cancer cells and tumour infiltrating lymphocytes were observed. Other components of immune response inhibition and tumour promotion are: Th17 cell population, M2 macrophage polarisation, the presence of myeloid derived suppressor cells (MDSCs) and a significantly elevated concentration of cytokines: TGF-b and IL-10 in the tumour microenvironment. The recognition of these mechanisms may have important therapeutic implications. Several types of agents which are capable of modulating the immune response have recently been used in many clinical trials conducted in lung cancer patients, some of them showing efficacy. Lung cancer immunotherapy has two main directions: the first goal is to improve the cytotoxic effect (for example by inhibition of CTLA-4, stimulation of dendritic cell function, inhibition of lymphocyte apoptosis), and the second way is the production of anti-cancer vaccines using known cancer antigens: MAGE A3, MUC1, EGF and TGF-b. Immunotherapy in lung cancer treatment has a character of personalised therapy - there is a need to specify the patient's immune status prior to treatment. The analysis of immune cells and mediators in the peripheral blood allows this, but the more valuable method seems to be bronchoalveolar lavage (BAL) examination with careful assessment of the tumour microenvironment.

Increase of Bcl-2/Bax ratio corelated with decrease of lymphocyte apoptosis: A study in the bronchiolus and lung of asthmatic mice

The failure of lymphocyte apoptosis is one of the factors causing chronic airway inflammation in asthma. Some studies indicate the role of Bcl-2 in inhibition of lymphocyte apoptosis, but still little research is on the role of Bax and its relationship to Bcl-2 in asthma. The purpose of this study was to prove the role of Bcl-2-lymphocyte in inhibition of lymphocyte apoptosis and decrease in Bax-lymphocytes in bronchiolus and lung of asthmatic mice. This study was a randomized control group design. Subjects were Balb/c mice which divided into 2 groups: non-asthma and asthma. Asthma group were sensitized with ovalbumin intraperitoneally on day 0 and 14, followed by inhalation every 2 to 3 days for 6 weeks. At week 8, all subjects terminated. Bcl-2 and Bax-lymphocytes expression were examined with immunohistochemical method, whereas apoptotic lymphocytes by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. Statistical analysis used was the independent sample t-test and regression analysis, with 95% confidence interval. Bronchial and lung specimens were obtained from 18 subjects (9 from each group). The ratio of apoptotic lymphocytes decreased in the asthma group (p = 0.003), Bcl-2-lymphocytes increased in the asthma group (p < 0.001), and Bax-lymphocytes decreased in the asthma group (p = 0.003). There was a strong negative correlation (r = -0.66, r2 = 0.43, p = 0.003) between the Bcl-2 and lymphocyte apoptosis. There was also a strong negative correlation (r = -0.56, r2 = 0.35, p = 0.009) between the ratio Bcl-2/Bax and lymphocyte apoptosis. However, no significant relationship was found between Bax-lymphocytes and lymphocyte apoptosis (r = 0.36, r2 = 0.13, p = 0.15). Increasing the ratio of Bcl-2/Bax inhibit lymphocyte apoptosis where Bcl-2 plays more role than Bax. Key words: Asthma, ovalbumin, lymphocyte apoptosis, Bcl-2, Bax.

Expression of caspase-8, receptor interacting protein and nuclear factor-kappaBp65 in oral lichen planus

To investigate the expressions of caspase-8, receptor interacting protein (RIP) and nuclear factor (NF)-kappaBp65 in oral lichen planus (OLP) and their relationship with cell apoptosis. Immunohistochemical technique with SP method was used to detect the expressions of caspase-8, RIP and NF-kappaBp65 in 30 OLP cases and 15 normal oral mucosa specimens. Terminal deoxynucleotidyl transferase-mediated nucleotide shift enzyme (TdT) mediated d-UTP end labeling (TUNEL) was used for detecting the cell apoptotic index (AI) in 15 OLP cases and 5 nomal oral mucosa specimens. Compared with the control group, the AI of epithelial cells (6.76 +/- 2.32) increased and the AI of lymphocytes (1.75 +/- 0.74)decreased in OLP (P < 0.01). The positive rate of caspase-8, RIP and NF-kappaBp65 of epithelial cells were 97% (29/30), 87% (26/30) and 93% (28/30) respectively, significantly higher in OLP than in normal control (P < 0.05). The positive rate of caspase-8, RIP and NF-kappaBp65 of lymphocytes were 100% (30/30, 90% (27/30) and 80% (24/30) respectively, significantly higher in OLP than in normal control (P < 0.01). A positive correlation was also observed between NF-kappaBp65 expression of lymphocytes and AI of epithelial cells. Accelerated apoptosis of the keratinocytes and inhibition of lymphocyte apoptosis may coexist and contribute to the formation and progression of OLP. The over expression of caspase-8, RIP and NF-kappaBp65 in OLP may play a role in the pathogenesis of OLP.

TARGETED DELIVERY OF siRNA TO CELL DEATH PROTEINS IN SEPSIS

Immune suppression is a major cause of morbidity and mortality in the patients with sepsis. Apoptotic loss of immune effector cells such as CD4 T and B cells is a key component in the loss of immune competence in sepsis. Inhibition of lymphocyte apoptosis has led to improved survival in animal models of sepsis. Using quantitative real-time polymerase chain reaction of isolated splenic CD4 T and B cells, we determined that Bim and PUMA, two key cell death proteins, are markedly upregulated during sepsis. Lymphocytes have been notoriously difficult to transfect with small interfering RNA (siRNA). Consequently a novel, cyclodextrin polymer-based, transferrin receptor-targeted, delivery vehicle was used to coadminister siRNA to Bim and PUMA to mice immediately after cecal ligation and puncture. Antiapoptotic siRNA-based therapy markedly decreased lymphocyte apoptosis and prevented the loss of splenic CD4 T and B cells. Flow cytometry confirmed in vivo delivery of siRNA to CD4 T and B cells and also demonstrated decreases in intracellular Bim and PUMA protein. In conclusion, Bim and PUMA are two critical mediators of immune cell death in sepsis. Use of a novel cyclodextrin polymer-based, transferrin receptor-targeted siRNA delivery vehicle enables effective administration of antiapoptotic siRNAs to lymphocytes and reverses the immune cell depletion that is a hallmark of this highly lethal disorder.

The role of IL-6 in inhibition of lymphocyte apoptosis by mesenchymal stem cells

Mesenchymal stem cells (MSCs) are widely distributed throughout the body. Despite intensive studies on the immunosuppressive effect of MSCs, little is known about whether MSCs affect lymphocyte apoptosis. We investigated the effect of MSCs on the spontaneous death of lymphocytes and found that MSCs inhibit the apoptosis of splenocytes and thymocytes as well as purified T and B cells. The protective effect of MSCs was absent when lymphocytes were not in contact with MSCs, indicating that the anti-apoptotic effect is exerted through direct interaction between MSCs and lymphocytes. Interestingly, this anti-apoptotic effect could be inhibited by neutralization of IL-6. Consequently, we found that the expression of IL-6 by MSCs was augmented by contact with lymphocytes. Taken together, these results demonstrate that IL-6 plays an important role in the inhibition of lymphocyte apoptosis by MSCs.

Inhibition of lymphocyte apoptosis by pancreatic stellate cells: impact of interleukin-15

There is growing evidence that pancreatic stellate cells (PSCs) produce cytokines and take part in the regulation of inflammatory processes in the pancreas. IL-15 inhibits apoptosis of various cell populations. This study was performed to investigate whether PSCs produce IL-15 and thereby can affect lymphocytes. Primary PSCs were isolated from the rat pancreas using density gradient centrifugation. mRNA expression of IL-15 was demonstrated by RT-PCR, and IL-15 protein was analyzed by immunoblotting. Lymphocytes obtained from rat mesenterial lymph nodes were cocultured with in vitro activated PSCs. Apoptosis has been quantified by the binding of annexin V-FITC with a flow cytometer. Proliferation was monitored using [3H]thymidine incorporation. PSCs express two splice variants of IL-15. The protein was detectable only in cell lysates but not in the cell culture supernatant. Cocultivation of lymphocytes with PSCs and IL-15 inhibited spontaneous lymphocyte apoptosis, and this effect was reduced by an anti-IL-15 antibody. Lymphocytes induced vice versa the proliferation and collagen production of PSCs. The inhibition of spontaneous lymphocyte apoptosis in cocultures with PSCs was at least partially mediated by cell-bound IL-15. This effect and the stimulation of PSCs by lymphocytes may lead to a circulus vitiosus, resulting in the persistence of inflammatory processes and the development of fibrosis during chronic pancreatitis.

Endotoxemia-induced Lymphocyte Apoptosis Is Augmented by a Hyperinsulinemic–Euglycemic Clamp

Sepsis and endotoxemia are associated with lymphocyte apoptosis. This has been regarded as harmful, contributing to further immune suppression in already immune-compromised patients. Because normalization of blood glucose improves outcome in critically ill patients, the authors hypothesized that one of the effects of insulin and normoglycemia would be inhibition of lymphocyte apoptosis. Therefore, in this experimental study in pigs, the authors examined the separate and combined effects of acute endotoxemia and a hyperinsulinemic-euglycemic clamp (HEC) on lymphocyte apoptosis. After 60 min of stabilization, 38 anesthetized and mechanically ventilated pigs (weight, 35-40 kg) were divided (by randomization performed before the experiment) into four groups and were then studied for 570 min. Group 1 received no intervention. Group 2 received a HEC (5 mm p-glucose, insulin infusion rate of 0.6 mU . kg (-1). min(-1)) for 570 min. Group 3 received a lipopolysaccharide infusion for 180 min. Group 4 was given a combination of a HEC and a lipopolysaccharide infusion. After the 570-min study period, the pigs were killed, and tissue was sampled from the spleen and frozen. In four sections of each sample, the apoptosis of B and T lymphocytes were analyzed using stereologic methods: The number of apoptotic B and T cells was estimated by fluorescence immunohistochemistry with anti-active caspase-3 and either anti-CD21 (B lymphocytes) or anti-CD3epsilon (T lymphocytes). The number of apoptotic B and T lymphocytes was then compared using two-way analysis of variance, and the interaction between endotoxemia and the clamp (hyperinsulinemia and euglycemia) was investigated. Endotoxemia induced apoptosis of B (P < 0.001) and T lymphocytes (P = 0.016) in the spleen, and this effect was independent of the clamp. The ratios of apoptotic cells in the spleen tissue of pigs with and without endotoxemia were 2.4 (confidence interval, 1.7-3.4) and 1.6 (confidence interval, 1.1-2.2) for B and T lymphocytes, respectively. Independent of endotoxin infusion, HEC increased the number of apoptotic lymphocytes (P = 0.029 and P = 0.038 for B and T lymphocytes, respectively). The ratios of the number of apoptotic spleen cells in pigs treated and not treated with HEC were 1.5 (confidence interval, 1.0-2.1) and 1.5 (confidence interval, 1.0-2.1) for B and T lymphocytes, respectively. In this porcine model, both endotoxemia and a HEC increased the number of apoptotic B and T lymphocytes in the spleen. Contrary to our hypothesis, lymphocyte apoptosis during acute endotoxemia was augmented by a HEC.

Improved survival in experimental sepsis with an orally administered inhibitor of apoptosis.

The pathophysiology of sepsis involves excessive lymphocyte apoptosis, which correlates with adverse outcomes, and disordered cytokine production, which may promote host injury. As the protease inhibitor (PI) class of antiretroviral agents is known to prevent apoptosis in vitro, we evaluated their effect on survival, lymphocyte apoptosis, and consequent cytokine production in mice with sepsis induced by cecal ligation and perforation. Mice pretreated with PIs have improved survival (67%; P<0.0005) compared with controls (17%) and a significant (P<0.05) reduction in lymphocyte apoptosis. Even mice receiving therapy beginning 4 h after perforation demonstrated improved survival (50%; P<0.05) compared with controls. PI therapy is also associated with an increase in the Th1 cytokine TNF-alpha (P<0.05) early in sepsis and a reduction in the Th2 cytokines IL-6 and IL-10 (P<0.05) late in sepsis; despite no intrinsic antibacterial effects, PI also reduced quantitative bacterial blood cultures. The beneficial effects of PI appear to be specific to lymphocyte apoptosis, as lymphocyte-deficient Rag1-/- mice did not experience benefit from treatment with PI. Thus, inhibition of lymphocyte apoptosis by PI is a candidate approach for the treatment of sepsis.

Links Between Type I Interferons and the Genetic Basis of Disease in Mouse Lupus

Systemic lupus erythematosus (SLE), like other autoimmune diseases, is a complex genetic trait with contributions from both major histocompatibility complex (MHC) genes and multiple non-MHC genes. Most of the contributing genes have yet to be identified. Studies of mouse models of lupus have provided important insight into the immunopathogenesis of lupus-like IgG autoantibody production and lupus nephritis, and genetic analyses of these mice are helping to unravel the complex and heterogeneous genetic basis of disease. Recent studies in both human SLE and mouse models of lupus have emphasized a potential role of type I interferons (IFN-α/β) in the initiation and perpetuation of disease. There is now increasing interest in genes that affect IFN-α/β expression–activity and IFN-regulated target genes that may be involved in the disease process. One example is interferon-inducible gene 202 (Ifi202), which has been identified as a major candidate susceptibility gene in the New Zealand hybrid model of lupus. Studies suggest that increased expression of this transcription factor leads to lupus through inhibition of lymphocyte apoptosis, although its effects on immune function are extremely complex and have yet to be fully defined. This review will focus on the genetic basis of disease in mouse lupus with a special emphasis on those genetic contributions that may affect IFN-α/β activity and those that may be target genes of IFN-α/β action.

In vitro suppression of thymocyte apoptosis by metal-rich complex environmental mixtures: potential role of zinc and cadmium excess

Excessive amounts of heavy metals (e.g. Zn, Cu, Mn, Cr) are accumulated in river bottom sediments (RBS), being available to humans and animals along food chains. Increased exposure of mammals to certain metals (Cr, Cu) induces immunosuppresion, due to DNA damage and decreased survival of lymphoid cells. By contrast, excess of Zn and Cd causes inhibition of apoptosis thus suggesting increased survival of genetically mutated cells and higher cancer risks in exposed populations. Rat thymic lymphocytes represent a well-established model for apoptosis testing. The primary goal of our study was to assess the degree of apoptosis modulation with a number of RBS extracts differing in their metal contents. A series of freshly deposited RBS was collected at nine sampling stations along the Elbe River. All sediments were rich in Fe, Mn and Zn. The contents of Cu, Cr, Ni, Cd, Hg, Pb and As were much lower and interrelated. The short-term cytotoxicity of aqueous sediment extracts was assessed, using the following criteria: total cell counts; incidence of apoptosis and necrosis (morphological detection by fluorescent microscopy); and nuclear chromatin decay (by DNA flow cytometry). RBS extracts produced both apoptosis and necrosis of thymocytes. High contents of zinc and other heavy metals in the samples correlated with decreased thymocyte apoptosis ( r=−0.543 to −0.608, P<0.01). The rates of thymocyte damage showed a distinct dependence on the time and region of sampling. Apoptosis modulation was also tested with pure salts of Mn(II), Zn(II), Cu(II), Cr(III) and Cd(II), at the test concentrations of 1, 10 and 100 μM. Cu(II) and Cr(III) proved to induce marked dose-related apoptosis, whereas Zn(II) ions caused significant suppression of apoptosis. These effects were similar to those trends observed with metal-rich sediments. In the present study, DNA flow cytometry proved to be a less sensitive index of cell death than morphological assay of apoptosis and/or necrosis. In summary, inhibition of lymphocyte apoptosis by RBS extracts and pure metals is associated with excess of zinc and, probably, cadmium. The proposed model of lymphoid cell apoptosis is a promising tool for screening cytotoxic effects of complex environmental samples.

Considering immunomodulatory therapies in the septic patient: should apoptosis be a potential therapeutic target?

Treatment of sepsis and septic shock remains a clinical conundrum. Recent prospective trials with anti-cytokine and anti-inflammatory therapies have shown only modest clinical benefit. The successful treatment of the patient with sepsis syndrome will likely require multi-modal therapies aimed at several of the immunological and physiological disturbances which are occurring simultaneously. Recent studies in experimental animals and critically ill patients have suggested that increased apoptosis of lymphoid organs and some parenchymal tissues may contribute to the immune suppression, anergy and organ system dysfunction. Therapies aimed at inhibiting lymphoid cell apoptosis may contribute to improved outcome, and should be considered in the treatment of hospitalized patients with sepsis syndromes. Although clinical trials with anti-apoptotic agents remain distant due in large part to technical difficulties associated with their administration and tissue targeting, inhibition of lymphocyte apoptosis may be an appropriate therapeutic target for the septic patient.

Inhibition of lymphocyte apoptosis

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