Apoptotic Lymphocytes Research Articles

Astrocyte-induced T cell elimination is CD95 ligand dependent

The brain has an intrinsic capacity to remove infiltrating T cells by inducing apoptosis. However, the pathways and cellular components driving this process are still under debate. Astrocytes seem to play an important role because they colocalize with apoptotic lymphocytes in vivo and induce apoptosis of transformed T cells in vitro. Since we previously demonstrated the expression of the death ligand CD95L (APO-1L/FasL) on astrocytes in the brain, we wanted to know whether nontransformed astrocytes induce cell death in nontransformed T cells, reflecting the in vivo situation and, if so, whether CD95/CD95 ligand interaction is important. T cell apoptosis measured by Annexin V binding and DNA fragmentation was significantly lower using CD95 ligand-deficient ( gld) astrocytes compared to nondeficient controls. Moreover, neutralizing anti-CD95 ligand antibody reduced astrocyte-induced T cell apoptosis. Thus, adult astrocytes are capable of inducing the apoptotic death of T cells by involving the CD95/CD95 ligand pathway without undergoing cell death in vitro. Since astrocytic end-feet contribute to the formation of the blood–brain barrier, this depletion mechanism may play an important role as the first line of defense in the brain.

Clearance of radiation-induced apoptotic lymphocytes: ex vivo studies and an in vitro co-culture model.

Lymphocytes are very sensitive to radiation. Our aim was to test the possibility of detecting apoptosis in lymphocytes as a potential short-term biomarker of ionizing radiation exposure. Our in vitro data confirmed the dose-time-effect relationships involved in radiation-induced apoptosis. The detection of in vivo induction of apoptosis in circulating lymphocytes after exposure of animals to radiation appears to depend critically on the technique used to measure apoptosis. Among the different techniques we investigated, mitochondrial modification was the most appropriate; they allowed establishment of dose-time-effect relationships when animals were observed for 72 h. A model of in vitro phagocytosis of apoptotic lymphocytes by macrophages was developed to mimic clearance of apoptotic cells occurring in vivo. Together, our data show that mitochondrial labeling may make it possible to detect ex vivo radiation-induced apoptosis of lymphocytes before macrophage ingestion occurs. We propose the measurement of apoptosis in lymphocytes as a potential short-term biomarker of ionizing radiation exposure.

Spontaneous tolerance: experience with the rat liver transplant model

Soluble major histocompatibility complex (MHC) class I antigens released from hepatocytes and the passenger leukocyte population of the liver allograft have both been considered as important contributors for spontaneous liver tolerance upon allogeneic transplantation into fully MHC-mismatched hosts. This study was conducted to delineate the role of “passenger leukocytes” (PL) as well as local intra-graft defence mechanisms of long-term accepted liver allografts in more detail. Orthotopic liver transplantation was performed in male inbred rats as follows ( n = 4–6): (i) Lewis (LEW; RT1. l) → LEW; (ii) DA (RT1. av1) → DA; (iii) DA → LEW; (iv) LEW → DA; (v) LEW (10-Gy whole body irridiation [WBI], d-7) → DA; (vi) LEW (10-Gy WBI, d-7) → LEW; (vii) LEW (10-Gy WBI, d-7) → LEW (parked for 36 hours) → DA; and (viii) LEW (10-Gy WBI, d-7) → DA (parked for 36 hours) → DA. The model specifically investigated the role of PLs as potential contributors to liver tolerance as WBI destroys this nonresident liver population. Characterization of Fas/FasL expression and the frequency of apoptotic cell death was performed by immunohistochemistry and TUNEL staining. Reverse transcriptase—polymerase chain reaction, by the use of Fas and FasL-specific cDNA probes, was performed on isolated hepatocytes of tolerized and rejected livers at various time points after transplantation. Animal survival in the various experimental groups was calculated using Kaplan-Meier survival statistics and related log-rank statistics as follows: (i) < 100 days; (ii) ≥ 100 days; (iii) > 100 days; (iv) 11.3 ± 1.0 day; (v) 9.0 ± 0.5 days; (vi) > 100 days; (vii) 5/6 > 100 days; and (viii) 8.0 ± 1.5 days. Immunohistochemistry revealed high numbers of proliferating cells in tolerized liver allografts. Apoptotic cell death of hepatocytes could be detected in both rejecting and to a lower extent in tolerized animals. Conversely, only tolerized but not rejected liver allografts revealed upregulation of FasL-expression on hepatic parenchymal cells from day 3 onwards. Irridiated LEW livers, in turn, loose their ability to survive in allogeneic DA hosts (group v) whereas they survive in syngeneic hosts (group vi), indicating that irridiation itself does not destroy the liver parenchyma. Reconstitution of irridiated LEW livers with syngeneic (group vii) but not with allogeneic (group viii) PLs restored tolerance induction. The underlying mechanisms of immune-privilege observed with liver allografts appear to share characteristics of clonal exhaustion suggesting that alloreactive lymphocytes are depleted by AICD via the FasL/Fas signal transduction pathway. The high frequency of apoptotic lymphocytes found in the portal tract of tolerized (but not rejected) LEW grafts supposes that functional FasL expression on graft hepatocytes mediates specific elimination of graft-directed effector lymphocytes. This mechanism constitutes peripheral deletion as one of the possible tolerogenic mechanisms involved. Chimerical liver grafts consisting of donor (LEW) parenchyma and host (DA) passenger leukocytes loose their tolerogenic capacity. In contrast, syngeneic reconstitution with LEW-PLs, restores liver graft acceptance upon transplantation into allogeneic DA hosts. This phenomenon is not relying on the induction of micro- or macrochimeric hosts, as no LEW PLs were found in spleen, thymus or the blood compartment from long-term surviving DA rats. Thus, non-resident liver cells contribute significantly to liver graft acceptance. Subsequently, liver tolerance appears to be mainly induced in the graft itself.

DNA fragmentation and morphological changes in apoptotic human lymphocytes

Cell suspensions enriched in cells at various stages of apoptosis were obtained by separation of irradiated human peripheral blood lymphocytes on density gradients at different post-irradiation times. The state of DNA fragmentation in the cells was determined by comet assay and pulsed field gel electrophoresis. The morphologically distinguishable features of apoptosis such as chromatin condensation and cell shrinkage correlated with discrete stages of DNA fragmentation. It was found that ⩾50 kbp fragmentation of DNA occurs already in cells of normal density whereas the subsequent DNA fragmentation onto fragments <50 kbp occurs in parallel with cell shrinkage and simultaneous increase in cell density.The observed stages of DNA fragmentation seem to be separated in time that could allow in case of abortive apoptosis formation of chromosomal aberrations.

Lymphocyte apoptosis and cell replacement in human liver allografts.

Apoptosis of graft-infiltrating T cells has been described after rodent liver transplantation. The aim of this study was to assess lymphocyte apoptosis in human allografts. Additionally, kinetics of leukocyte turnover were studied to determine whether apoptotic cells were likely to be of donor or recipient origin. Liver biopsy specimens (n=36) taken between days 3 and 1855 were stained with terminal deoxynucleotidyl transferase dUTP nick end-labeling and anti-CD3 to detect apoptotic lymphocytes. Renal allograft and hepatitis C biopsy specimens served as controls. Donor cell turnover was studied in sex-mismatched grafts using Y-chromosome in situ hybridization to detect recipient cells and double immunostaining for leukocyte phenotyping. T-cell apoptosis was prominent in hepatic sinusoids (72% of biopsy specimens) as early as day 3. It ranged from 0% to 18.2% of CD3+ cells (mean 5.28+/-0.82%) and persisted for >14 days, including time points >1 year. There was no difference between biopsy specimens with or without rejection (6.34+/-1.14% and 4.61+/-1.13%, P=NS). Apoptotic cells in portal tracts were less frequent (33% of biopsy specimens) and less abundant (1.13+/-0.36%, P<0.0001). No lymphocyte apoptosis was seen in renal allograft biopsy specimens or hepatitis C biopsy specimens, indicating that it is a distinctive feature of the liver allograft. Persisting lymphocyte apoptosis even after donor lymphocytes had been replaced suggests that recipient lymphocyte deletion must occur. Donor Kupffer cells persisted for many months. Our results suggest that the sinusoidal microenvironment promotes recipient lymphocyte apoptosis, which may account for the improved outcome of liver grafts compared with other organ allografts.

Apoptotic lymphocytes induced by surgical trauma in dogs.

An investigation was made of the population and function of lymphocytes in canine peripheral blood, in animals with or without laparotomy under inhalation anesthesia. Fourteen healthy beagles were allocated to two experimental groups: laparotomy (group A) and without laparotomy (group B). Induction of anesthesia in both groups was carried out with an intravenous injection of thiopentone, and was maintained by isoflurane inhalation. Ten blood samples, consisting of 10 ml of venous blood withdrawn by venipuncture into syringes containing 200 units of preservative-free heparin, were taken, from before anesthesia to the 28th postoperative day. The blood samples were collected, and the number of lymphocytes, the lymphocyte subpopulations, the proportion of apoptotic lymphocytes, and plasma cortisol level were measured, and the blastoid transformation of lymphocytes was observed. Lymphopenia was observed in both groups after anesthesia. Flow cytometry indicated a greater reduction in the proportion of T lymphocytes than of B lymphocytes. Blast transformation was also depressed in both groups. Progression of apoptosis after anesthesia was demonstrated in both groups, with a higher percentage of apoptotic cells being observed in group A at 12 h after anesthesia (28.5 +/- 3.2% by TUNEL assay). Plasma levels of cortisol were elevated to a greater extent in group A at the end of anesthesia (10.3 +/- 0.8 microg/dl) than in group B (7.8 +/- 1.9 microg/dl). These results indicate that surgical trauma concomitant with anesthesia could impair immunocompetence by reducing the number and function of lymphocytes.

THE EFFECT OF PARTICIPATION IN AN ULTRA-MARATHON ON LYMPHOCYTE APOPTOSIS

Lymphocyte damage has been reported following both treadmill runs to exhaustion and marathon participation. Evidence is derived from rat studies of thymic involution and thymocyte apoptosis and human studies of single strand DNA breaks and lymphocyte apoptosis. Human studies are limited and little is known of the effect of exercise of different intensity and duration on lymphocyte apoptosis. PURPOSE: To investigate the degree of lymphocyte apoptosis following participation in a 90 km ultra marathon. METHODS: Seven healthy participants in the 2000 Comrades Marathon were studied. There were 6 males and 1 female. The average age was 34 ± 6 years, height 1.72 ± 3.1 cm and weight 74 ± 4.8 Kg. Venous blood samples were taken in the afternoon, 48 hours before the race, and at the same time of day, 24 and 48 hours after the race. Full blood counts and differential counts were performed in the hospital hematology department. CD3, CD4, CD8, CD19 and CD56 counts were performed using flow cytometric methods and lymphocyte apoptosis was determined using the Annexin V kit. RESULTS: All subjects finished the race in times ranging from 7h28min to 10h13min. Apoptosis was demonstrated in 10.9 ± 6.6% of lymphocytes before the race, in 26.5 ± 3.4% one day after the race and in 17.9 ± 3.2% of lymphocytes 2 days after the race. ANOVA with post hoc testing showed significant increases in apoptosis one and two days after the race (p < 0.01) and a significant difference between the percentages of apoptotic lymphocytes in the post race samples. Both CD4 and CD8 counts increased after the race, with the CD4:CD8 ratio decreasing from 2.1 ± 1.1 before the race to 2.0 ± 0.7 1 day after and 1.8 ± 0.6, 2 days after. CONCLUSIONS: Participation in an ultra marathon is associated with apoptosis of lymphocytes and apoptosis continues for at least 48 hours after the event. The signal inducing exercise evoked lymphocyte apoptosis requires further investigation, as does the mechanism by which the signaling lasts for up to 48 hours.

Induction of Biologically Active Antineutrophil Cytoplasmic Antibodies by Immunization with Human Apoptotic Polymorphonuclear Leukocytes

Translocation of intracellular components to the cell surface during the priming or apoptosis of polymorphonuclear leukocytes (PMN) is an important mechanism for interaction of antineutrophil cytoplasmic antibodies (ANCA) with these antigens. To test the capacity of apoptotic PMN to trigger production of ANCA, six groups of mice were immunized with either live or apoptotic lymphocytes, or with live, apoptotic, formalin-fixed, or lysed PMN. Mice immunized with both live and apoptotic neutrophils developed high titers of antibodies which gave a granular cytoplasmic immunofluorescent pattern. These antibodies were specific for lactoferrin and myeloperoxidase. Following a second intravenous infusion of apoptotic PMNs, mice developed anti-PR3 antibodies. Vasculitis lesions were not found in mice which developed ANCA. The ANCA-containing IgG fraction induced superoxide production by human PMNs. These results support the hypothesis that neutrophil-specific antigens presented on the cell membranes of apoptotic PMN may induce ANCA in the proper conditions.

The tumor-associated antigen, RCAS1, can be expressed in immune-mediated diseases as well as in carcinomas of biliary tract

Background/Aims: RCAS1, which is recognized by the specific antibody 22-1-1, was first identified as a tumor-associated antigen in gynecological carcinomas. RCAS1 is the ligand of a putative receptor present on lymphocytes, the expression of which is enhanced by lymphocyte activation. RCAS1 inhibits the growth of receptor-bearing cells and induces apoptotic death. Here we examined RCAS1 expression in biliary diseases. Methods: RCAS1 expression was immunohistochemically examined on tissue samples. Apoptotic death was analyzed by DNA fragmentation detection method. RCAS1 production by cell lines was investigated by flow cytometry, enzyme-linked immunosorbent assay, and reverse transcription–polymerase chain reaction. Results: All cholangiocarcinoma cell lines examined clearly expressed RCAS1 at both the protein and RNA level. Immunohistochemically, RCAS1 was expressed in a high percentage of biliary adenocarcinomas (85.9% of intrahepatic cholangiocarcinomas, 96.4% of extrahepatic cholangiocarcinomas and gallbladder carcinomas). Apoptotic tumor-infiltrating lymphocytes could be found in these specimens. RCAS1 expression was frequently detected also in biliary epithelial cells in cases of immune-mediated cholangitis (74.2% in primary biliary cirrhosis, 66.6% in graft-versus-host disease), although the staining pattern for RCAS1 was different compared with cancer cells. Conclusions: RCAS1 is highly expressed not only in cancer cells but also in non-tumor bile duct cells subjected to immune attack.

A small-volume technique for simultaneous immunophenotyping and apoptosis detection in rat whole blood by four-color flow cytometry.

Cell permeabilization for the detection of intracellular molecules by flow cytometry is usually incompatible with whole blood. This article describes a new technique for the simultaneous detection of surface antigens and DNA content in rat whole blood. In 20 microl of rat whole blood, DNA staining is obtained by permeabilization of cells using a standard red blood cell lysing reagent (Erythrolyse). Immunophenotyping and apoptosis detection by flow cytometry are achieved by using a combination of three surface markers (CD3, CD4, and CD8alpha) and a DNA binding dye (TO-PRO-3). After a 24-h incubation of whole blood with 1 microM dexamethasone, apoptotic lymphocytes were clearly distinguishable from normal lymphocytes by their reduced size and DNA content. The dexamethasone-induced percentage of apoptotic cells was 58.9 +/- 4.6 for CD4+ and 77.4 +/- 2.9 for CD8+ T cells, compared with 12.6 +/- 2.7 for CD4+ and 17.2 +/- 3.5 for CD8+ T cells in the absence of dexamethasone (data from 10 animals with duplicate samples). We have developed a new technique to permeabilize nucleated cells in microsamples of rat whole blood. The methodology allows simultaneous immunophenotyping and apoptosis detection in rat whole blood.

Prolonged exercise, lymphocyte apoptosis and F2-isoprostanes.

Exercise induces a post-exercise decline in the number of circulating lymphocytes. The aim of the present study was to investigate whether strenuous exercise induces lymphocyte apoptosis and generation of reactive oxygen species. Eleven healthy male subjects exercised for 2.5 h on a treadmill. Apoptotic lymphocytes were defined by being annexin positive and 7-aminoactinomycin-D negative. Measurement of F(2)-isoprostanes was used as a marker of oxidant stress in vivo. An increase (60%, P<0.05) in the percentage of apoptotic circulating lymphocytes was found 2 h post-exercise, whereas the total number of apoptotic cells did not change in relation to exercise. The concentration of plasma F(2)-isoprostanes increased approximately 1.6-fold in response to exercise, but declined towards pre-exercise values within the 1st h of recovery. The plasma concentrations of adrenaline, noradrenaline and cortisol increased during exercise. In conclusion, the results of the present study demonstrate that even in a study design in which high levels of apoptosis-inducing factors are generated, such as cortisol and isoprostanes, lymphocyte apoptosis does not contribute to post-exercise lymphocytopenia.

Relationship between lymphocyte apoptosis and endotoxin translocation after thermal injury in rats.

To investigate the relationship between lymphocyte apoptosis in peripheral blood, spleen and mesenteric lymph nodes(MLN) and endotoxin translocation after thermal injury in rats. In a Wistar rat model inflicted with 30% TBSA III degree scalding, serum LPS levels in portal vein and vena cava were quantified by tachypleus amebocyte lysate (TAL) technique. The analysis of peripheral blood lymphocyte was employed in in situ Cell Death Detection Kit and evaluated by flow cytometry. Apoptotic lymphocytes in paraffin-embedded spleen and MLN sections were examined by histologic analysis, in situ deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and peroxidase (POD) staining. The images were taken by Cooldccd camera system, and the count and optical density value (transmission light) of apoptotic lymphocytes were analyzed with software Spot and Imagine proplus 4.10a(IPP4.10a). In the period of 3 to 48 postburn hours (PBHs) serum LPS level (x10(3) EU.L(-1)) in portal vein (2.11+/-0.02, 5.66+/-0.20, 3.70+/-0.22, 2.56+/-0.28, 0.90+/-0.11) was higher than that in vena cava (0.63+/-0.01, 1.53+/-0.18, 0.83+/-0.32, 0.52+/-0.12, 0.23+/-0.02, P<0.01), but both increased sharply in postburn rats (P<0.01) and reached a peak at 6 PBH. Analysis of apoptotic lymphocytes showed that the proportion (%) of postburn apoptotic cells was much higher than that in healthy rats (8.34+/-1.53, 8.13+/-1.81, 20.77+/-3.94, 23.90+/-3.92, 11.23+/-1.35 and 13.26+/-2.09 at 3, 6, 12, 24, 48 and 72 PBH, respectively, vs 3.99+/-1.72, P<0.01), especially after 6 PBH. The concentrations of lymphocytic apoptosis at 12 and 24 PBH were markedly higher than that at other time points. Meantime, few apoptotic lymphocytes were found in normal MLN, but increased postburn obviously (3+/-1 vs 546+/-83, 285+/-39, 149+/-30, 58+/-10, 36+/-11 and 33+/-9 in turn, P<0.01), especially at 3 PBH, whereas apoptotic lymphocytes were concentrated in splenic cortex before the burn and decreased obviously during 72 PBHs (499+/-186 vs 12+/-8, 19+/-15, 12+/-7, 100+/-15, 123+/-25 and 226+/-26 in turn, P<0.01) though a slight rise was found in the medulla after 24 PBH. Optical density of apoptotic lymphocytes was significantly reduced in spleen in the 24 PBHs and raised in MLN during 48 PBHs than that prior to the burn, respectively. Gut-origin LPS is a major cause of endotoxemia taken place early in rats following severe thermal injury and could induce extensive lymphocyte apoptosis in blood and MLN, which suggests an immunosuppression state could follow the initial injury and favores a septic state based on apoptotic mechanism.

Large quantity of ribosomal RNA exists extracellularly in mouse spleen.

When BALB/c mouse spleens were gently homogenized in saline, the resultant supernatant (without cells and tissue debris) contained significant amount of 28S and 18S ribosomal RNA, reaching up to 70% of the total spleen RNA. Haemoglobin assays indicated that less than 15% of the spleen cells were lysed during the homogenization process, indicating that the majority of the spleen 'supernatant RNA' was from the extracellular space of the organ rather than released by the splenocytes as a consequence of grinding. Quantitative RNA analysis showed that the ratio of spleen supernatant RNA/total RNA of BALB/c mice was inversely correlated with age (from approximately 70% at 3 weeks to 45% at 6 months), but that of BXSB mice (an animal model for systemic lupus erythematosus) remained at about 70% irrespective of age. Methyl Green-Pyronin Y staining of paraffin sections of mouse spleen revealed that extracellular RNA was distributed mainly in the sinuses of the organ. Culture supernatants of apoptotic splenocytes contained significant amounts of RNA, suggesting that the extracellular RNA in the spleen might have come from apoptotic lymphocytes. This is supported by the fact that 'thymus supernatant' also contained significant amount of RNA. A possible correlation between spleen extracellular RNA and autoimmune diseases is discussed.

In vitro cytotoxicity assay to screen compounds for apoptosis-inducing potential on lymphocytes and neutrophils.

In vitro cytotoxicity assay to screen compounds for apoptosis-inducing potential on lymphocytes and neutrophils was investigated. Mouse, rat, dog, and human whole blood were incubated for 4 and 6 hr with actinomycin D, camptothecin, cortisone acetate, cycloheximide, doxorubicin, etoposide, 5-FU, mitomycin C and puromycin. Apoptotic lymphocytes and neutrophils were counted. All test compounds induced in vitro apoptosis of lymphocytes and/or neutrophils, but there were different potencies among the test compounds and there were also species differences in susceptibility. To investigate the in vivo effects of etoposide and cycloheximide which induced apoptosis of rat lymphocytes and that of rat lymphocytes and neutrophils, respectively, in in vitro assay, rats were intravenously administered either etoposide at 12.5, 25 or 50 mg/kg or cycloheximide at 1.25, 2.5 or 5 mg/kg. Etoposide caused decreases of circulating lymphocytes at 3 hr after administration in a dose-dependent manner, -16, -25 and -51%. Although cycloheximide caused neither decreased lymphocyte nor neutrophil counts, apoptosis in 30% of neutrophils was observed in rats receiving 5 mg/kg at 3 hr after administration. Etoposide at 50 mg/kg and cycloheximide at 5 mg/kg caused lymphocyte apoptosis in the spleen, thymus, mesenteric lymph nodes, bone marrow, and Peyer's patch from 1 to 6 hr after administration, with the maximum changes at 3 hr. In addition to apoptosis of these organs, cycloheximide at 5 mg/kg caused apoptosis of polymorphonuclear cells in the lamina propria of the small intestine. Therefore, it was found that the changes seen in the in vivo experiments considerably reflected the changes seen in the in vitro experiments. From these results, apoptosis is probably one of the major mechanisms for leukocyte toxicity induced by cytotoxic compounds, and the in vitro assay to screen compounds for acute apoptosis-inducing potential on lymphocytes and neutrophils would be useful as a primary screening method for animal toxicity studies. It may also be useful for risk assessments in advance of clinical trials.

Annexin V expression in apoptotic peripheral blood lymphocytes: an electron microscopic evaluation.

Loss of plasma membrane asymmetry, resulting in the exposure of phosphatidylserine (PS), is considered to be an 'early' event in apoptosis. It is generally accepted to precede nuclear condensation, independent of the apoptosis inductive agent. In the present study we focus on 2 apoptotic parameters: PS exposure in comparison with morphological alterations. Peripheral blood lymphocytes were irradiated in vitro (5 Gy Co-gamma-rays) or incubated with staurosporine (1 microM, 6 hours). PS exposure was measured flow cytometrically using FITC-labelled annexin V, combined with PI. Morphological alterations were evaluated by electron microscopy (EM). Results are based on 3 independent experiments. For the irradiated lymphocytes the amount of viable cells (annexin V-/PI-) as scored by flow cytometry was comparable or slightly lower than the number of viable cells as scored by EM (75% compared to 79%). However, for the staurosporine treated lymphocytes only about 24% of the cells were designated as viable by EM, whereas by flow cytometry about 65% of the cells were annexin V-/PI-. Examination by EM showed about 40% cells with a morphology distinct from that of a normal viable cell, but without the clear-cut characteristics of apoptotic cells. Time studies revealed that these cells went into apoptosis after prolonged incubation times up to 18 hours. Application of biotinilated annexin V for EM detection with gold-conjugated anti-biotin, showed that only clear-cut apoptotic, apoptotic necrotic and oncotic cells showed the gold-label at their membranes. Cells that could be detected under the EM as 'non-viable' but without the clear-cut characteristics of apoptotic cells, were not labelled. Data indicate that, dependent on the apoptosis inductive mechanism, morphological alterations can occur before PS exposure.

Resistance of lymphocytes to Fas-mediated apoptosis in Behçet's disease and Vogt-Koyangi-Harada syndrome

Background : Behçet's disease and Vogt-Koyanagi-Harada (VKH) syndrome are characterized by a severe intraocular inflammation with a long course and recurrent episodes. Activated autoreactive lymphocytes are considered to be responsible for the development of this inflammation. These lymphocytes may also be involved in the perpetuation and recurrence of this inflammation if they do not properly go into apoptosis. To investigate this hypothesis, a study was carried out to evaluate the resistance of lymphocytes to apoptosis in these two uveitis entities. Methods : Blood samples were obtained from 17 patients with Behçet's disease, 15 patients with VKH syndrome, and 11 healthy individuals. Mononuclear cells were isolated by centrifugation with Ficoll-Paque and cultured in RPMI 1640 with or without phytohemag-glutinin (PHA) for 9 h at 37°C in an atmosphere with 5% CO 2. The obtained cells were incubated with anti-Fas antibody for 8 h at 37°C in an atmosphere with 5% CO 2. The cells were stained with annexin V/propidium iodide and finally subjected to flow cytometry. Results : A significantly lower percentage of apoptotic lymphocytes after PHA stimulation was noted in Behçet's disease (19.7 ± 4.1%) and VKH syndrome (20.4 ± 6.9%) than in controls (26.1 ± 7.3%). The percentage of apoptotic lymphocytes without PHA stimulation also tended to be lower in the patients with Behçet's disease (12.6%) and with VKH syndrome (12.8%) than in controls (14.6%), although the difference was not significant. Conclusion : Lymphocytes in patients with either Behçet's disease or VKH syndrome are relatively resistant to apoptosis mediated by anti-Fas antibody. These apoptosis-resistant, or long-lived, lymphocytes may be involved in the chronic and recurrent intraocular inflammation seen in these patients.

Nivalenol-induced apoptosis in thymus, spleen and Peyer's patches of mice

ICR:CD-1 male mice were orally administered with Nivalenol(NIV) at the dose levels of 5, 10 and 15 mg/kg body weight, and examined at 12, 24 and 48 hours after inoculation (HAI), respectively, to elucidate the process of development of apoptosis in the thymus, spleen and Peyer's patch. There were no signs of clinical disorders and no changes in body and organ weights until 48 HAI except for that the thymus weight significantly decreased at 48 HAI. Immunohistochemically, the number of apoptotic lymphocytes evaluated by in situ detection for fragmented DNA showed a dose-dependent increase at 12 HAI in both the thymus and the Peyer's patch, while it became to increase at 24 HAI in the spleen. Dead lymphocytes in the thymus, spleen and Peyer's patch showed ultrastructural characteristics of apoptosis. Moreover, the DNA ladder was first detected by agarose gel electrophoresis at 12 HAI in the thymus of 15 mg/kg-group. The results clearly indicate that NIV is able to induce apoptosis in the lymphoid tissues of mice.

Proinflammatory response during Ebola virus infection of primate models: possible involvement of the tumor necrosis factor receptor superfamily

Ebola virus (EBOV) infections are characterized by dysregulation of normal host immune responses. Insight into the mechanism came from recent studies in nonhuman primates, which showed that EBOV infects cells of the mononuclear phagocyte system (MPS), resulting in apoptosis of bystander lymphocytes. In this study, we evaluated serum levels of cytokines/chemokines in EBOV-infected nonhuman primates, as possible correlates of this bystander apoptosis. Increased levels of interferon (IFN)-α, IFN-β, interleukin (IL)-6, IL-18, MIP-1α, and MIP-1β were observed in all EBOV-infected monkeys, indicating the occurrence of a strong proinflammatory response. To investigate the mechanism(s) involved in lymphoid apoptosis, soluble Fas (sFas) and nitrate accumulation were measured. sFas was detected in 4/9 animals, while, elevations of nitrate accumulation occurred in 3/3 animals. To further evaluate the potential role of these factors in the observed bystander apoptosis and intact animals, in vitro cultures were prepared of adherent human monocytes/macrophages (PHM), and monocytes differentiated into immature dendritic cells (DC). These cultures were infected with EBOV and analyzed for cytokine/chemokine induction and expression of apoptosis-related genes. In addition, the in vitro EBOV infection of peripheral blood mononuclear cells (PBMC) resulted in strong cytokine/chemokine induction, a marked increase in lactate dehydrogenase (LDH) activity, and an increase in the number of apoptotic lymphocytes examined by electron microscopy. Increased levels of sFAS were detected in PHM cultures, although, <10% of the cells were positive by immunohistochemistry. In contrast, >90% of EBOV-infected PHM were positive for tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) by immunohistochemistry, RNA analysis, and flow cytometry. Inactivated EBOV also effected increased TRAIL expression in PHM, suggesting that the TNF receptor superfamily may be involved in apoptosis of the host lymphoid cells, and that induction may occur independent of viral replication. In further studies with infected PHM, expression of MHC II was remarkably suppressed after 6 days, an additional correlate of immunological dysregulation. In conclusion, our findings suggest that infection of mononuclear phagocytes is critical, triggering a cascade of events involving cytokines/chemokines and oxygen free radicals. It is the consequence of these events rather than direct viral infection that results in much of the observed pathology. Identification of cytokine/chemokine, nitric oxide, and reactive oxygen species involvement in the observed filoviral pathogenesis may lend insight into the rational design of therapeutic countermeasures of filoviral pathogenesis.

Proapoptotic and antiapoptotic markers in cutaneous T-cell lymphoma skin infiltrates and lymphomatoid papulosis.

In cutaneous T-cell lymphoma (CTCL) lesions, both reactive T cells and malignant T cells intermingle. The disease progression is mostly slow. Recent evidence suggests that even if clinical remission is reached, malignant cells persist and a relapse follows sooner or later. To wha extent tumour cell apoptosis occurs in the skin lesions either due to the reactive T cells or t therapeutic efforts is not known. To determine the extent of tumour cell apoptosis and the expression of proapoptotic an antiapoptotic markers in serial skin lesion samples from patients with CTCL, and to compare th findings with those in patients with lymphomatoid papulosis (LyP). Thirty-four skin samples were obtained from 12 patients with CTCL at the time o diagnosis and at a mean of 1.6, 3 and 6 years later. The patients received psoralen plus ultraviolet (PUVA), electron beam or cytostatic treatments. In addition, fresh post-treatment samples fro three patients with CTCL undergoing PUVA therapy were obtained. For comparison, skin biopsies o five patients with LyP were studied. Immunohistochemical demonstration of the expression of th following markers was performed on formalin-fixed skin sections: Fas (CD95), Fas ligand (FasL) bcl-2, granzyme B, the tumour-suppressor protein PTEN and the effector caspase, caspase-3. Th malignant cells were identified morphologically, and apoptotic cells were identified with th terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling method on parallel sections. In untreated CTCL lesions, apoptotic lymphocytes were extremely rare, and no increase in the number of apoptotic cells was observed after any of the treatments used. In LyP, apoptotic cell were more frequent, comprising on average 5% of the infiltrate. The apoptosis-associated marker Fas, FasL, caspase-3 and granzyme B were expressed by morphologically neoplastic cells in CTCL and by large atypical cells in LyP, with no significant differences. However, only a few reactive cell in CTCL infiltrates expressed granzyme B while about 10% of the corresponding cells were positive in LyP. The expression of antiapoptotic bcl-2 was more frequent in CTCL than in LyP, while PTE expression was high in both instances. The number of bcl-2 + cells tended to decrease after therapy When comparing the findings between the first and the last samples, a decrease in the number of bcl-2+ cells and an increase in Fas+ cells was associated with disease progression, despite therapy, while the opposite was true for remissions. Apoptosis was found to be a rare event in CTCL lesions irrespective of precedin therapy During patient follow-up, no significant differences in the expression of apoptotic marker was observed while in most cases a lower level of antiapoptotic bcl-2 expression was observed after all types of therapies and in association with disease progression when compared with high expression in the untreated lesions. The absence of apoptosis and high expression of bcl-2 together with a low expression of apoptosis-inducing granzyme B in the reactive lymphocytes in CTC could explain the chronic nature of the disease and the poor response to therapy, while th more frequent occurrence of granzyme B and apoptosis together with a lower level of expressio of bcl-2 by the large atypical cells in LyP could contribute to the favourable outcome of the latter.

Fas expression on peripheral blood lymphocytes in systemic lupus erythematosus (SLE): relation to lymphocyte activation and disease activity.

Levels of apoptotic lymphocytes have been found to be increased in SLE and persistence of apoptotic cells has been associated with autoantibody production. Increased lymphocyte Fas (CD95) expression due to lymphocyte activation may account for increased susceptibility to Fas-mediated apoptosis in SLE. Flowcytometry was performed to evaluate membrane expression of Fas in combination with the activation markers CD25, HLA-DR and CD38 on, respectively, CD4+, CD8+ and CD19+ lymphocytes of SLE patients with inactive (n = 20) and with active disease (n = 13). SLEDAI-scores were calculated. Healthy volunteers (n = 14) served as controls. Percentages of CD4+ T-cells expressing CD25 and CD19+ B-cells expressing CD38 were increased in patients with active disease compared to controls (P = 0.03, P = 0.04, respectively). In contrast to CD4+ and CD8+ cells, percentages of CD19+ cells expressing Fas were increased in SLE patients with active disease (P = 0.0002 vs controls). In these patients percentages of cells double positive for both CD38 and Fas were increased compared to patients with inactive disease (P = 0.006) and controls (P = 0.0007). Percentages of CD19+ cells expressing Fas correlated with SLEDAI-scores. In SLE patients, percentages of Fas-expressing B-lymphocytes are increased, are related to the state of lymphocyte activation, and correlate to disease activity. Increased Fas expression results in a higher susceptibility for Fas-mediated apoptosis, which might contribute to the increased levels of apoptotic lymphocytes in SLE patients.