Early antigen elimination by cytotoxic T cells results in diminished cellular immune responses to allogeneic tumor

Previous reports have suggested that repeated alloantigenic challenge increases humoral responses to alloantigens, but may cause decreasing cellular responses. We stimulated BALB/c (H-2^d) mice with intraperitoneal EL-4 tumor (H-2^b) and serially assessed cytotoxic responses in spleen and peritoneal lymphocytes using ⁵¹Cr-labeled EL-4 target cells. We observed that cytotoxicity generated in the spleen of nonimmune BALB/c mice was much greater than that in immunized mice; similar peak responses were generated in peritoneal lymphocytes in normal and immunized hosts. Complement-mediated cytotoxicity was not necessary for diminished splenic responses in hyperimmune hosts, for the same phenomenon was seen in the Hzl anti-BL/6 system which is free of humoral responses. Irradiation (2000 rad) of the EL-4 tumor challenge prevented tumor cell proliferation and markedly reduced splenic responses in nonimmune mice. We suggest that cytotoxic cells suppressed further generation of cyto-toxicity; by effecting an early elimination of tumor inoculum, tumor proliferation was abrogated and dose of cellular antigen was, consequently, markedly reduced.     

Radical mediators and mitogen-activated protein kinase signaling in oxygen-dependent radiosensitivity of human tumor cell lines

Oxygen enhancement of tumor radiosensitivity is attributed to DNA damage by reactive oxygen species. The mechanism remains unclear but may involve mitochondria as major sources of oxygen and nitrogen radicals as well as central effectors of energy homeostasis and apoptosis. Here we used dihydrorhodamine and 2',7'-dichlorodihydrofluorescein to compare mitochondrial and total cell generation, respectively, of reactive oxygen or nitrogen species in cells irradiated at 5 Gy. Irradiation in the presence of oxygen selectively stimulated mitochondrial radical production in HeLa and MeWo cells, but in MCF7 cells radical production was more generalized. In all three cell lines oxygen impaired cell proliferation as measured by resazurin reduction 7 days after irradiation. Antioxidants N-acetylcysteine, ascorbic acid, and melatonin largely prevented dye oxidation during normoxic irradiation yet had no effect on oxygen-dependent irradiation injury. However, NO synthase inhibitor N(G)-monomethyl-L-arginine protected HeLa and MCF7 though not MeWo cells, consistent with their different levels of constitutive NO generation. SB203580 inhibition of p38 MAPK appreciably protected HeLa and marginally protected MCF7 cells against oxygen-dependent irradiation injury, while the less specific JNK/SAPK inhibitor SP600125 and ERK inhibitor U0126 had no effect. None of the inhibitors affected MeWo radiosensitivity. Therefore oxygen-enhanced radiosensitivity in these tumor cell lines does not depend on extensive production of oxygen radicals and is cell-type dependent. NO mediates oxygen-dependent injury in HeLa and MCF7 cells, by p38-dependent and MAPK-independent mechanisms, respectively. In MeWo cells this oxygen-enhanced radiosensitivity is independent of both NO and MAPK signaling.     

Immunological Properties of TtT/M-87 Cell Line Established from Murine Pituitary Tumor-Associated Macrophages

TtT/M-87 cell is a macrophage cell line established from thyrotropic pituitary tumor tissues in mouse. In this paper, we report the immunological properties of M-87 cells as a model of tumor-associated macrophage. Contrasting with resident peritoneal macrophages, M-87 cells constitutively secreted small but significant amounts of TNF-α and IL-1α, which were detectable in both biological assays (cytotoxic activity for L929 and co-mitogenic activity for Con A-induced T cell proliferation, respectively) and ELISA, and produced larger amounts of these cytokines upon stimulation with LPS. They expressed MHC class II molecules on their cell surface without stimulation by IFN-γ. The accessory or antigen-presenting cell activity in antibody-producing response of spleen lymphocytes to sheep red blood cells was shown to be much higher in M-87 cells than normal peritoneal macrophages. In addition, when normal spleen lymphocytes were cultured with allogeneic tumor cells, such as EL-4 and S-180, in the presence of M-87 cells, lymphocytes reactive to stimulator cells were activated to manifest inhibitory effect on the tumor cell growth and also to manifest specific cytotoxic effect on the allogeneic tumor cells. These results show that M-87 cells derived from tumor-associated tissue are activated macrophages and that they are inhibitory to tumor cell growth and augmentative in the induction of T-cell-mediated immune responses.     

Depletion of macrophages expressing I-J antigen results in efficient generation of alloreactive cytotoxic T lymphocytes

The role of suppressor macrophages (S-M phi) produced during generation of cytotoxic T lymphocytes (CTL) stimulated with allogeneic lymphocytes was investigated. Splenic CTL from C3H/He mice (H-2k) were generated by in vivo immunization and subsequent in vitro stimulation by splenic lymphocytes from C57B1/6 mice (H-2b) in mixed lymphocyte reaction (MLR). In addition to in vitro standard 51Cr release assay, the CTL activity was mainly measured in vivo using the Winn assay against EL-4 thymoma cells in B6C3F1 mice (H-2b/k). In mice injected with CTL plus EL-4 cells survival rate was 20% compared with no survival of mice treated with normal spleen cells plus EL-4 cells. The antitumor activity of the CTL was significantly increased when immunized mice were treated with a 5 mg/kg ip dose of indomethacin at the time of immunization (80% survival). Macrophages were depleted from spleen cells of immunized mice by plastic adherence or carbonyl-iron treatment, replaced with an equivalent number of M phi from normal mice, and then introduced into a 5-day MLR. When the antitumor activity of the cells isolated from this MLR was measured in the Winn assay, 90-100% survival in EL-4-bearing mice was observed. In contrast, none of the mice inoculated with EL-4 alone and 20% of the mice that received CTL obtained after alloimmunization followed by MLR in addition to EL-4 survived. These results of CTL activity were confirmed by in vitro cytotoxicity tests. When the M phi isolated from spleens of immunized mice were analyzed for I-Jk antigen expression, a 2.5-fold increase was detected, compared with splenic M phi obtained from normal C3H/He mice. In contrast, Ia and I-Ak antigen expression was equivalent in M phi isolated from normal or immunized C3H/He mice. When immune spleen cells were treated with anti-I-Jk antiserum followed by complement and then, subjected to the MLR, the antitumor activity of CTL was significantly enhanced (80% survival). However, treatment of these cells with anti-I-Ak antiserum and complement did not alter CTL activity. These data suggest that the increase of S-M phi expressing I-Jk+ antigen to be induced during alloimmunization results in suppression of allospecific CTL-generation in MLR.     

Different induction of adaptive response to ionizing radiation in normal and neoplastic cells

Since the beneficial effects of low-dose radiation (0.01 Gy) are usually observed in normal cells, we investigated whether the adaptive response was induced by low-dose radiation in neoplastic cells of different origin as well as in normal cells. Cell lines used in this experiment were as follows: mouse lymphocytes (NL); L929 cells established from mouse connective tissue; primary mouse keratinocytes (PK); line 308 from mouse papilloma; X-ray sensitive lymphoma cells, L5178Y-S and EL-4 cells from mouse lymphoma. The adaptive response was determined by cell survival and apoptosis. The involvement of apoptosis in the adaptive response was examined by ELISA and TUNEL assay. Adaptive response was induced by pretreatment with low-dose radiation of 0.01 Gy in normal cells such as NL, L929, and PK, but not in L5178Y-S, EL-4, and line 308 cells. In addition, the reduction of apoptosis by pretreatment with low-dose radiation was observed in NL, L929, and PK, but not in L5178Y-S, EL-4, and line 308 cells. These results suggested that the adaptive response could be induced by pretreatment with low-dose radiation and the phenomena were observed in normal cells, not in neoplastic cells. In addition, pretreatment with low-dose radiation reduced apoptosis, suggesting that an anti-apoptotic pathway may be involved in the adaptive response. This revised version was published online in July 2006 with corrections to the Cover Date.     

Modulation of EL-4 mouse lymphoma cell proliferation by macrophages and tumor related factors

It is well known that macrophages play an important role in the control of tumor growth. This control may be the result of a direct action of macrophages or mediated by several biologically active products or factors elaborated by these and other cell populations. Our studies on the proliferation of a murine T-cell lymphoma (EL-4) showed that the treatment of the ascitic fluid (from the peritoneum of EL-4 bearing mice) with carbonyl iron resulted in a depletion of phagocytes concomitant with a significant increase of [3H] thymidine uptake by EL-4 cells. Further, the growth of EL-4 cells cultured in semisolid agar was significantly inhibited by an underlayer of large quantities of macrophages both from normal and EL-4 bearing mice as well as when cultured in the presence of PGE2. The underlayer of tumor macrophages P388 D1 resulted in an increase of the EL-4 cell growth. Also, conditioned media obtained from in vitro liquid cultures of EL-4 cells and L 1210 cells (B-lymphoma) produced a remarkable inhibition of the in vitro cloning capacity and [3H] thymidine uptake by EL-4 cells. These data support the hypothesis that different factors from normal and hemopoietic tumor cells may control the tumor growth and point out that self-produced factors may modulate the proliferation of tumor cells.     

Three different signals are required for the induction of cytolytic T lymphocytes from resting precursors

The requirement for the signals in induction of cytolytic T lymphocytes (CTL) has been investigated. C57BL/6 X CBA/T6 F1 spleen cells stimulated with the lectin leukoagglutinin (L-A) failed to show CTL activity in a PHA-facilitated assay, although L-A-activated splenic T cells were able to respond to T cell growth factor (TCGF). Concanavalin A (Con A) on the other hand was able to induce cytolytic activity from CTL-P, as well as to render splenic T cells responsive to TCGF. Furthermore, L-A-activated splenic T cells could generate cytolytic activity upon subsequent culture in secondary mixed leukocyte culture supernatant (2 degrees MLC SN). In contrast, EL-4-derived SN (EL-4 SN) was unable to induce cytolytic activity from L-A-activated spleen cells. In addition, proliferation of L-A-activated spleen cells cultured in EL-4 SN was similar to those cultured in 2 degrees MLC SN. Nonactivated spleen cells were totally unresponsive to both SN in proliferation and generation of CTL. Analysis of T cell clones for the production of a factor necessary for induction of cytolytic activity revealed that both cytolytic and noncytolytic T cell clones were able to produce a factor(s) for the generation of cytolytic activity from L-A-activated T cells. On the other hand, SN from certain antigen-stimulated T cell clones produced factors capable of inducing cytocytic activity by L-A-activated T cells only in the presence of EL-4 SN. Neither EL-4 SN nor cloned T cell SN alone had such a capacity. The nature of the necessary lymphokines in the SN from the clone cells or from the EL-4 is unknown. In the case of the EL-4 SN, it is not known whether the presence of TCGF plays a role or whether that role is perhaps more differentiative than proliferative. This study provides evidence that the induction of CTL from CTL-P can be dissociated into activation, which is required to render T cells responsive to second signals, and differentiation, which is mediated by two different factors.     

Mouse mammary tumor virus-related sequences in mouse lymphocytes are inducible by 12-O-tetradecanoyl phorbol-13-acetate

cDNA libraries from EL-4 cells treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) were screened for TPA-inducible sequences by differential hybridization. The most abundant inducible species was a sequence similar to that of mouse mammary tumor virus (MMTV). Induction of the mRNA corresponding to the MMTV-related sequences was already evident 30 min after TPA treatment, whereas the maximum accumulation occurred after 20 h of exposure to TPA. TPA also increased levels of MMTV-related RNA in the normal spleen cells of BALB/c and C57BL/6 mice. The level of RNA expression corresponding to MMTV-related sequences, however, was markedly elevated in EL-4 cells as compared with normal spleen cells. Southern blots of EL-4 cell DNA showed that the MMTV-related sequences were inserted into multiple locations of the EL-4 genome. Sequence analysis revealed that the MMTV-related cDNA clones included a part of the env gene and the right long terminal repeat of MMTV. However, the cDNA sequences were substantially different from published MMTV proviral sequences, most notably because of a contiguous deletion of 491 base pairs in the open reading frame within the U3 region.     

Apoptosis development pathways in human lymphocytes induced by UV light and reactive oxygen species

We have studied alterations in the structural state of DNA, the level of membrane Fas-receptor expression, functional activity of caspase-3, the concentration of Ca²⁺, p53 and cytochrome c proteins in human lymphocyte cells in the dynamics of apoptosis, induced by UV light (240–390 nm) at doses of 151, 1510, and 3020 J/m² and reactive oxygen species (ROS): superoxide anion radical, hydroxyl radical, hydrogen peroxide, and singlet oxygen. It was established that UV light and ROS induce lymphocyte DNA fragmentation after the incubation of a modified cell for 20 h. It was shown that in 1–5 h after UV light and ROS exposure on lymphocytes, an increase is observed in the level of membrane death Fas-receptors as compared to intact cells. Enhancement was revealed in the functional activity of lymphocyte caspase-3 4 h after the generation of singlet oxygen, hydroxyl radical, and the addition of hydrogen peroxide, as well as 8 and 24 h and 6 and 8 h of UV irradiation of cells at doses of 151 and 1510 J/m², respectively. Using the DNA comet approach, it was revealed that DNA damage (single-stranded breaks) appears approximately 15–20 min after UV irradiation of lymphocytes at doses of 1510 and 3020 J/m² and the addition of hydrogen peroxide at a concentration of 10⁻⁶ mol/L (comets of the C1 type) and reaches its maximum 6 h after cell modification (comets of the C2 and C3 types). Six hours after exposure of lymphocytes to hydrogen peroxide and UV light at doses of 1510 and 3020 J/m², it was established that the p53 level increased in the investigated cells. It was established that under UV light exposure and exogenous generation of reactive oxygen species, the increase in the calcium level in lymphocyte cytoplasm is determined by Ca²⁺ efflux from the intracellular depots as a result of activation of the components of the phosphoinositide information transmission mechanism to a cell. A hypothesis was proposed on the correlation between changes in the calcium level and initiation of programmed cell death in human lymphocytes after UV light and ROS exposure. It was concluded that the lead role is played by receptor-mediated (Fas-dependent) caspase and p53-dependent pathways in the development of lymphocyte apoptosis induced by exposure to UV light at doses of 151 and 1510 J/m² and reactive oxygen metabolites. A scheme is presented which considers possible intracellular events leading to apoptotic death of lymphocytes after UV irradiation.     

Radiation-induced bystander effect in healthy G(o) human lymphocytes: biological and clinical significance

To study the bystander effects, G(0) human peripheral blood lymphocytes were X-irradiated with 0.1, 0.5 and 3 Gy. After 24h, cell-free conditioned media from irradiated cultures were transferred to unexposed lymphocytes. Following 48 h of medium transfer, viability, induction of apoptosis, telomere shortening, reactive oxygen species (ROS) levels and micronuclei (after stimulation) were analyzed. A statistically significant decrement in cell viability, concomitant with the loss of mitochondrial membrane potential, telomere shortening, increases in hydrogen peroxide (H(2)O(2)) and superoxide anion (O(2)(-)) with depletion of intracellular glutathione (GSH) level, and higher frequencies of micronuclei, were observed in bystander lymphocytes incubated with medium from 0.5 and 3 Gy irradiated samples, compared to lymphocytes unexposed. Furthermore, no statistically significant difference between the response to 0.5 and 3 Gy of irradiation in bystander lymphocytes, was found. However, when lymphocytes were irradiated with 0.1 Gy, no bystander effect with regard to viability, apoptosis, telomere length, and micronuclei was observed, although a high production of ROS level persisted. Radiation in the presence of the radical scavenger dimethyl sulfoxide (DMSO) suppressed oxidative stress induced by 3 Gy of X-rays with the effective elimination of bystander effects, suggesting a correlation between ROS and bystander signal formation in irradiated cells. The data propose that bystander effect might be mostly due to the reactions of radiation induced free radicals on DNA, with the existence of a threshold at which the bystander signal is not operative (0.1 Gy dose of X-rays). Our results may have clinical implications for health risk associated with radiation exposure.     

Augmentation of the antitumor effect of adoptive immunotherapy by in vivo sensitization of EL-4 lymphoma and pre-treatment with sizofiran

The antitumor effects of adoptive immunotherapy using LAKcells treated with sizofiran (SPG) following in vivoantigen sensitization with EL-4 lymphoma (EsLAK),comparing nonsensitized LAK cells (sLAK), were studied inmice with intraperitoneal implantation of EL-4 lymphoma.EL-4 cells treated with Mitomycin C (100 g /ml) wereintroduced by inoculation into the peritoneum of C57BL/6mice for antigen sensitization. Four days later, SPG (100g) was intramuscularly injected. Three days after SPG administration, mononuclear cells obtained from the spleen were prepared for LAK cells (EsLAK). The following resultswere obtained: 1) The survival period was significantlygreater in the sLAK and EsLAK groups than in the controlgroup. The survival period in the EsLAK group wassignificantly greater than that in the sLAK group. 2) Thenumber of EL-4 cells in the peritoneal exudate cells 11days postimplantation was lowest in the EsLAK group, andthe number of lymphocytes including LGL was largest in theEsLAK group, compared with the sLAK group and the controlgroup. 3 ) The EsLAK cells showed significantly moreenhanced cytotoxic activity against EL-4 than the sLAKcells. 4) Histopathological findingsof metastatic lesions of the liver and spleen stained by HE11 days postimplantation showed less infiltrating tumorcells and more lymphocytic infiltrations in the sLAK andEsLAK groups compared with the control group. Theseresults suggest that induction of LAK cells byadministration of SPG to lymphocytes treated by in vivosensitization with tumor antigen increasesthe efficacy of adoptive immunotherapy.     

软枣猕猴桃总黄酮对V79细胞的辐射防护作用

目的:研究软枣猕猴桃总黄酮的辐射防护活性。方法:以V79细胞辐射损伤模型为体外验证模型,利用回流醇提法提取的软枣猕猴桃总黄酮于辐射前处理细胞,细胞经8 Gy60Coγ射线辐射后,用多功能酶标仪检测细胞的存活率,用流式细胞仪检测细胞内活性氧及细胞凋亡率的变化。结果:用50～200μg/mL的软枣猕猴桃总黄酮于辐射前给药细胞,进行24 h培育,能抑制细胞内因辐射引起的活性氧上升,从而提高细胞存活率。结论:软枣猕猴桃总黄酮能清除细胞内由辐射产生的活性氧,降低细胞凋亡率,达到保护V79细胞的效果。     

巴西莓和诺尼果粉对C57BL/6J小鼠抗8Gy 剂量辐射的初步研究

目的:比较研究巴西莓果粉Herbal Clean Energy和诺尼果粉Noni GIA通过消除自由基、降低造血细胞凋亡等作用对8Gy大剂量γ线照射后小鼠活存的影响。方法:将C57BL/6J小鼠随机分组、每组雌雄各半,单一果粉在相同灌胃剂量下采用照前灌胃10天、照后灌胃10天以及照前照后灌胃10天三种灌胃方式,首先观察了小鼠在用钴60γ线8Gy致死剂量照后40天活存率;其次在上述相同条件处理下,照后10天对小鼠外周血白细胞计数、CD4+和CD8+淋巴细胞类型、活性氧、骨髓造血细胞凋亡率等分析。结果:生理盐水灌胃组C57BL/6J小鼠受8Gy照射在第18天全部死亡(n=20,下同),死亡率100%,而照前10天灌胃后照射8Gy试验组:巴西莓和诺尼果粉组照后40天活存10/20只,诺尼果粉组第40天活存9/20,巴西莓果粉组第40天活存8/20。在照后灌胃的组别中,诺尼果粉组第40天活存7/20,巴西莓和诺尼果粉组第40天活存4/20只,巴西莓果粉组第40天活存2/20。照射前后单独或联合灌胃两种果粉组外周血白细胞均有升高,而骨髓造血细胞凋亡降低,而且果粉灌胃小鼠外周血Th/Tc比率同对照组相比明显保持于正常值范围。红细胞和血小板数据无明显变化,活性氧含量则呈现无规律表现。结论:巴西莓和诺尼果粉对小鼠抗辐射有预防作用,其细胞学表现为保持造血增殖能力、降低造血细胞凋亡,并维持Th/Tc免疫平衡;显示果粉对保持造血和免疫能力是提高抵抗辐射损伤、提高活存的基础。同时表明,服用巴西莓果粉和诺尼果粉,对人体防止辐射损伤有预防作用。     

Effect of cytotoxic lymphocytes obtained by in vitro immunization with syngeneic lymphoma cells on pluripotent hemopoietic cells

The possibility of the presence of leukemia-associated antigens on pluripotent hemopoietic cells was studied with the aid of immune lymphocytes, cytotoxic against mouse syngeneic lymphoma cells. Cytotoxic lymphocytes were obtained during immunization in vitro of C57BL/6 mouse splenocytes by syngeneic T lymphoma EL-4 cells in the presence of interleukin-2. Specific cytotoxic activity of immune lymphocytes as regards EL-4 cells was not blocked by addition of normal bone marrow cells. Incubation of the bone marrow with immune killers did not lead to a decrease in the number of colony-forming units in the spleen. It was shown that using cytotoxic lymphocytes the total killing of lymphoma cells might be achieved in a mixture of bone marrow and lymphoma cells, whereas pluripotent precursor cells might be retained.     

Triazole analog 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol induces reactive oxygen species and autophagy-dependent apoptosis in both in vitro and in vivo breast cancer models

Autophagy is considered as an important cell death mechanism that closely interacts with other common cell death programs like apoptosis. Critical role of autophagy in cell death makes it a promising, yet challenging therapeutic target for cancer. We identified a series of 1,2,3-triazole analogs having significant breast cancer inhibition property. Therefore, we attempted to study whether autophagy and apoptosis were involved in the process of cancer cell inhibition. The lead molecule, 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol (T-12) induced significant cell cycle arrest, mitochondrial membrane depolarization, apoptosis and autophagy in MCF-7 and MDA-MB-231 cells. T-12 increased reactive oxygen species and its inhibition by N-acetyl-l-cysteine protected breast cancer cells from autophagy and apoptosis. Autophagy inhibitor, 3-methyladenine abolished T-12 induced apoptosis, mitochondrial membrane depolarization and reactive oxygen species generation. This suggested that T-12 induced autophagy facilitated cell death rather than cell survival. Pan-caspase inhibition did not abrogate T-12 induced autophagy, suggesting that autophagy precedes apoptosis. In addition, T-12 inhibited cell survival pathway signaling proteins, Akt, mTOR and Erk1/2. T-12 also induced significant regression of tumor with oral dose of as low as 10 mg/kg bodyweight in rat mammary tumor model without any apparent toxicity. In presence of reactive oxygen species inhibitor (N-acetyl-l-cysteine) and autophagy inhibitor (chloroquine), T-12 induced tumor regression was significantly decreased. In conclusion, T-12 is a potent inducer of autophagy-dependent apoptosis in breast cancer cells both in vitro and in vivo and can serve as an important lead in development of new anti-tumor therapy.     

Gliotoxin enhances radiotherapy via inhibition of radiation-induced GADD45a, p38, and NFkappaB activation

The purpose of the study was to elucidate the mechanism underlying the enhancement of radiosensitivity to 60Co gamma-irradiation in human hepatoma cell line HepG2 pretreated with gliotoxin. Enhancement of radiotherapy by gliotoxin was investigated in vitro with human hepatoma HepG2 cell line. Apoptosis related proteins were evaluated by Western blotting. Annexin V/PI and reactive oxygen species (ROS) were quantified by Flow Cytometric (FACS) analysis. Gliotoxin (200 ng/ml) combined with radiation (4 Gy) treated cells induced apoptosis. Cells treated with gliotoxin (200 ng/ml) prior to irradiation at 4 Gy induced the expression of bax and nitric oxide (NO). The gliotoxin-irradiated cells also increased caspase-3 activation and ROS. Gadd45a, p38, and nuclear factor kappa B (NFkappaB) activated in irradiated cells was inhibited by Gliotoxin. Specific inhibitors of p38 kinase, SB203580, significantly inhibited NFkappaB activation and increased the cytotoxicity effect in cells exposed to gliotoxin combined with irradiation. However, SB203580 did not suppress the activation of Gadd45a in irradiated cells. Gliotoxin inhibited anti-apoptotic signal pathway involving the activation of Gadd45a-p38-NFkappaB mediated survival pathway that prevent radiation-induced cell death. Therefore, gliotoxin, blocking inflammation pathway and enhancing irradiation-induced apoptosis, is a promising agent to increase the radiotherapy of tumor cells.     

Translocation of cytoplasmic HSP70 onto the surface of EL-4 cells during apoptosis

Heat shock proteins (HSPs) are involved in a variety of intracellular processes and can have both pro- and anti-apoptotic action. However, little is known about the role of HSPs in the progression of apoptosis. Translocation of HSPs to the surface of apoptotic cells is a previously observed phenomenon demonstrating participation of these proteins in execution of the terminal stages of apoptosis. In a previous study we showed that development of EL-4 lymphoma cell apoptosis in vitro is accompanied by elevation of surface HSP expression. In this study we used this model to analyse the relationship of HSP70 expression and its translocation to the cell surface during apoptosis with some key intracellular events. Our data demonstrate a synchronization of surface and intracellular HSP70 expression with bcl-2 expression, intracellular reactive oxygen species (ROS) concentration and caspase-3 activity. A maximum level of surface and intracellular HSP70 expression was observed at an irreversible phase of EL-4 cell apoptosis after mitochondrial potential depolarization. In addition, an enhancement of the relative level of cytoplasmic HSP70 translocation to the cell surface was concomitant with EL-4 cell apoptosis. However, the size of surface and intracellular pools of HSP70, increasing for initial and intermediate stages of cell death, decreased at the terminal phase of apoptosis. Western blot analysis of HSP70 in conditioned supernatant obtained from EL-4 cell tissue showed that the observed decrease of HSP70 cell content might be due to surface HSP70 shedding into the intercellular space.     

Nicotinamide riboside intervention alleviates hematopoietic system injury of ionizing radiation-induced premature aging mice

Radiotherapy destroys cancer cells and inevitably harms normal human tissues, causing delayed effects of acute radiation exposure (DEARE) and accelerating the aging process in most survivors. However, effective methods for preventing premature aging induced by ionizing radiation are lacking. In this study, the premature aging mice of DEARE model was established after 6 Gy total body irradiation (TBI). Then the therapeutic effects and mechanism of nicotinamide riboside on the premature aging mice were evaluated. The results showed that 6 Gy TBI induced premature aging of the hematopoietic system in mice. Nicotinamide riboside treatment reversed aging spleen phenotypes by inhibiting cellular senescence and ameliorated serum metabolism profiles. Further results demonstrated that nicotinamide riboside supplementation alleviated the myeloid bias of hematopoietic stem cells and temporarily restored the regenerative capacity of hematopoietic stem cells probably by mitigating the reactive oxygen species activated GCN2/eIF2α/ATF4 signaling pathway. The results of this study firstly indicate that nicotinamide riboside shows potential as a DEARE therapeutic agent for radiation-exposed populations and patients who received radiotherapy.     

Interleukin 2 enhancement of lymphokine secretion by T lymphocytes: analysis of established clones and primary limiting dilution microcultures

The effect of exogenous interleukin 2 (IL 2) on lymphokine production by T lymphocytes was examined in two systems: the secretion of macrophage-activating factor (MAF) and interferon (IFN) by cloned long-term T cell lines, and a limiting dilution system for estimating the frequency of precursors of MAF-secreting cells in normal spleen. An IL 2-containing, MAF- and IFN-free supernatant from the EL-4 thymoma (EL-4 SN) significantly enhanced release of MAF and IFN by mitogen- or antigen-stimulated, cytolytic or noncytolytic T lymphocyte clones directed against alloantigens or Moloney leukemia virus-associated antigens. Highly purified IL 2 produced equivalent enhancement as EL-4 SN in cultures of alloreactive clones stimulated with concanavalin A. Kinetics experiments showed that EL-4 SN increased both the rate and duration of MAF release by T cell clones. EL-4 SN also increased MAF production when added during restimulation of limiting dilution cultures of positively selected Lyt-2+ and Lyt-2- C57BL/6 splenic T lymphocytes activated against DBA/2 alloantigens. This enhancement resulted in a threefold increase in the apparent precursor frequency of MAF-secreting cells among Lyt-2+ lymphocytes, but did not affect the frequency among Lyt-2- cells. Additional analysis indicated that average MAF production in cultures of Lyt-2-+ cells was sixfold lower than in cultures of Lyt-2- cells, and hence that EL-4 SN allowed detection of a significant proportion of Lyt-2+ cell cultures secreting low levels of MAF. Under these improved conditions, the MAF assay detected the majority of responding Lyt-2+ and Lyt-2- lymphocytes.     

Studies on the induction and expression of T cell-mediated immunity. IV. Non-overlapping populations of alloimmune cytotoxic lymphocytes with specificity for tumor-associated antigens and transplantation antigens.

Two non-overlapping populations of alloimmune cytotoxic T cells with specificity for tumor-associated antigens (TAA) and for histocompatibility antigens (H-2) were characterized by two independent methods. The heterogeneity of cytotoxic cells was demonstrated in spleen cells derived from BALB/c (H-2d) mice sensitized to EL-4 (H-2b) tumor and from C57BL/6 (H-2b) mice sensitized to G-35 (H-2d) tumor cells. Adsorption of immune lymphocytes on monolayers prepared with cells bearing the sensitizing H-2 antigens abrogated the in vitro cell-mediated cytotoxicity (CMC) directed against 51Cr-labeled normal target cells (spleen cells or ConA-activated spleen blasts), whereas significant cytolytic activity to the corresponding 51Cr-tumor cells was still retained. Likewise, in competitive inhibition assays, CMC to 51 Cr-tumor target cells was only partially inhibited by unlabeled normal cells, whereas CMC to 51Cr-normal target cells was completely abrogated. These results suggested that alloimmune cytotoxic lymphocytes are heterogeneous and can be subdivided into two independent populations of restricted specificity. Several experiments suggested that the effector cell population directed against TAA can no longer elicit a graft-vs-host (GVH) reaction in vivo. This was demonstrated by adoptive transfer into lethally-irradiated allogeneic recipients of cytotoxic or primed spleen cells fractionated on host target cell monolayers. Furthermore, these results demonstrated that both effector cells and memory cells possess high affinity binding receptors to corresponding H-2 antigens. The potential use of fractionated immune lymphocytes sensitized to tumor allografts in adoptive immunotherapy is discussed.