Temporally distinct response of irradiated normal human fibroblasts and their bystander cells to energetic heavy ions

Ionizing radiation-induced bystander effects have been documented for a multitude of endpoints such as mutations, chromosome aberrations and cell death, which arise in nonirradiated bystander cells having received signals from directly irradiated cells; however, energetic heavy ion-induced bystander response is incompletely characterized. To address this, we employed precise microbeams of carbon and neon ions for targeting only a very small fraction of cells in confluent fibroblast cultures. Conventional broadfield irradiation was conducted in parallel to see the effects in irradiated cells. Exposure of 0.00026% of cells led to nearly 10% reductions in the clonogenic survival and twofold rises in the apoptotic incidence regardless of ion species. Whilst apoptotic frequency increased with time up to 72 h postirradiation in irradiated cells, its frequency escalated up to 24h postirradiation but declined at 48 h postirradiation in bystander cells, indicating that bystander cells exhibit transient commitment to apoptosis. Carbon- and neon-ion microbeam irradiation similarly caused almost twofold increments in the levels of serine 15-phosphorylated p53 proteins, irrespective of whether 0.00026, 0.0013 or 0.0066% of cells were targeted. Whereas the levels of phosphorylated p53 were elevated and remained unchanged at 2h and 6h postirradiation in irradiated cells, its levels rose at 6h postirradiation but not at 2h postirradiation in bystander cells, suggesting that bystander cells manifest delayed p53 phosphorylation. Collectively, our results indicate that heavy ions inactivate clonogenic potential of bystander cells, and that the time course of the response to heavy ions differs between irradiated and bystander cells. These induced bystander responses could be a defensive mechanism that minimizes further expansion of aberrant cells.     

Generation of reactive oxygen species and radiation response in lymphocytes and tumor cells

Several types of lymphoid and myeloid tumor cells are known to be relatively resistant to radiation-induced apoptosis compared to normal lymphocytes. The intracellular generation of reactive oxygen species was measured in irradiated spleen cells from C57BL/6 and BALB/c mice and murine tumor cells (EL-4 and P388) by flow cytometry using dichlorodihydrofluoresceindiacetate and dihydrorhodamine 123 as fluorescent probes. The amount of reactive oxygen species generated per cell was low in the tumor cells compared to spleen cells exposed to 1 to 10 Gy of gamma radiation. This could be due to the higher total antioxidant levels in tumor cells compared to normal cells. Further, the changes in mitochondrial membrane potential and cytoplasmic Ca2+ content were appreciable in lymphocytes even at a dose of 1 Gy. In EL-4 cells, no such changes were observed at any of the doses used. About 65% of spleen cells underwent apoptosis 24 h after 1 Gy irradiation. However, under the same conditions, EL-4 and P388 cells failed to undergo apoptosis, but they accumulated in G2/M phase. Thus the intrinsic radioresistance of tumor cells may be due to a decreased generation of reactive oxygen species after irradiation and down-regulation of the subsequent events leading to apoptosis.     

Mechanism of tolerance to VI-antigen of Salmonella typhi induced by cyclophosphane

High dose Vi-antigen treatment and injection of cyclophosphamide 46 to 48 hours later induced in mice a state of immunological unresponsiveness remaining stable in adoptive transfer. Only low amounts of the antigen were revealed in the blood and spleen of tolerant animals 2 to 3 weeks after the tolerogenic treatment. No T-suppressors were found in the spleen of tolerant mice--the cells of tolerant mice failed to suppress the immune response of normal lymphocytes when transferred together to the irradiated recipients, or to induce tolerance in normal mice. Normal spleen cells restored partially the immune responsiveness in tolerant animals. The results obtained suggest that cyclophosphamide tolerance was due to deletion or the long-term inactivation of the immunocompetent cells.     

Regulation of cell-mediated cytotoxicity. III. Synergism of cortico-resistant with anti-thymocyte serum-resistant splenic T cells in the generation of cytotoxic lymphocytes in graft-vs-host reactions.

Lethally irradiated F1 hybrid mice were given an i.v. injection of parental strain spleen cells. Six days later, their spleen cells were used as the effector cells to measure the in vitro cell-mediated cytotoxicity (CMC) of the parental cells. The treatment of the donors with hydrocortisone resulted in a marked decrease of the capacity of their spleen cells to produce a CMC reaction, whereas the treatment with antithymocyte serum (ATS) resulted in an almost complete loss of such activity. The mixing of spleen cells from hydrocortisone-treated parental donors with the spleen cells from ATS-treated parental donors before injection resulted in a synergistic amplification of the cytotoxic response. The anti-Thy-1 serum treatment of either spleen cell population abolished the synergism completely. These results indicate that cortico-resistant T cells act as precursors of cytotoxic lymphocytes and that ATS-resistant T cells produce an amplification of their reaction.     

Macrophage-mediated in vitro sensitization of lymphocytes

Summary Antigen-fed macrophages were able to induce specific sensitization of unprimed syngeneic lymphocytes in vitro. The sensitized lymphocytes caused specific injury to target cells that carried the relevant antigens. In the present study, we investigated the in vivo activity of lymphocytes sensitized by antigen-fed macrophages. Mouse spleen cells were sensitized by macrophages that had been exposed to the radiation leukemia virus (RadLV). The sensitized lymphocytes, which were enriched for T-cells, were injected to syngeneic normal recipients and 4 days later the mice were challenged with RadLV-induced lymphoma cells. By following tumor growth and survival of mice, we have found that the sensitized lymphocytes protected the recipient mice against lymphoma development if injected 4 days before the tumor cells. The protective activity of the sensitized lymphocytes was radioresistant, but they could not protect irradiated hosts. It is suggested that macrophagemediated in vitro sensitization of lymphocytes induces initiator cells that can protect the recipient host by recruitment of a defensive immune response.     

p53-Mediated gene activation in mice at high doses of chronic low-dose-rate γ radiation

The time course of the changes in the expression of p53-mediated genes in vivo after high doses of chronic low-dose-rate γ radiation remains unclear. Here we analyzed peripheral blood cell counts and the expression of p53-mediated genes in the spleens of mice chronically irradiated at low dose rate (0.0167 Gy/h) for 1-40 days. Low-dose-rate irradiation induced p53-dependent chronic decreases in white blood cell (WBC) counts in p53 wild-type mice. Upregulation of p53-mediated genes by low-dose-rate radiation was confirmed in the whole spleen cells from the p53 wild-type mice, while suppressed gene expression was observed in the spleen cells of p53-deficient mice. The expression of p21 and Bax in radiosensitive cells such as T and B lymphocytes from low-dose-rate irradiated mice at 10, 20, and 40 days were increased, although that of Mdm2 in both the lymphocytes was decreased at 20 and 40 days. Moreover, spleen weights for low-dose-rate irradiated mice were decreased at 20 and 40 days. Thus downregulation of Mdm2 in both T and B lymphocytes by low-dose-rate radiation may cause higher p53 activation; further, higher p53 expression may determine the radiosensitivity and cause a reduction in the spleen weights in low-dose-rate irradiated mice. These results indicate that p53 may be chronically activated by low-dose-rate radiation.     

A requirement for helper T cells in the induction of delayed-type hypersensitivity

This paper describes a model system for studying the role of helper T cells in the induction of delayed-type hypersensitivity (DTH). Cyclophosphamide- (CP) treated mice sensitized with antigen 3 days later develop high levels of delayed-type immunity; however, DTH cannot be demonstrated in mice that are sensitized with antigen 1 day after drug treatment. The inability to respond to antigen 1 day after CP treatment can be restored if either normal or low-dose primed spleen cells are transferred at the time of sensitization. Although irradiated (1500 rad) normal spleen cells are unable to restore DTH, such treatment has no effect on the primed spleen cell population. The lymphocytes responsible for restoring the DTH response were identified as T cells, in that treatment with anti-Thy-1.2 serum and C abrogated their effect. Furthermore, restoration of the DTH response was dependent on the presence of antigen at the time of lymphocyte transfer; irradiated primed cells could not transfer DTH alone. The DTH effector cells in reconstituted mice were identified as originating from the host and not from the transferred cell population. This was accomplished by using anti-H-2 serum to identify the source of the DTH effector cells after transferring parental (H-2b) irradiated primed spleen cells into CP-treated F1 mice (H-2b,k). Thus, the irradiated transferred cells are behaving as helper T cells and promoting the development of DTH effector cells in the host.     

Release of mtDNA from mitochondria and activation of its replication in tissues of irradiated mice

A nessessary condition for normal functioning of mitochondria is the maintenance of certain numbers of intact mtDNA molecules. In the present study, we investigasted changes in the number of mtDNA copies in brain and spleen cells of mice subjected to irradiation. For the first time, we observed the irradiation-induced output of mtDNA fragments into brain and spleen cell cytosol. In the cytosol of these cells, examined in mice 5 h after 5 Gy irradiation, 1841 h.p. mtDNA fragments were detected able to persist for at 3 weeks. In addition, larger fragments of mtDNA (10,090 b.p.) were detected in the cytosol of brain cells of irradiated mice. The occurrence of mtDNA fragments in the cytosol of brain cells is accompanied with an increase in the number of mtDNA copies in the mitochondrial matrix. The induction of mtDNA replication in brain cells of irradiated animals may be considered as a compensatory reaction in response to mtDNA damage. A sharp decrease in the amount of mtDNA copies in the mitochondrial matrix of spleen cells on the first day after irradiation may be considered as apoptosis development. However, the compensatory reaction in brain cells was also noticed but in later terms.     

Increased apoptosis in lamina propria B cells during polymicrobial sepsis is FasL but not endotoxin mediated

Recent studies from our laboratory demonstrated that mucosal lymphoid tissue such as Peyer's patch cells and lamina propria (LP) B lymphocytes from mice shows evidence of increased apoptosis after sepsis that is associated with localized inflammation/activation. The mechanism for this is poorly understood. Endotoxin as well as Fas/Fas ligand (FasL) have been shown to augment lymphocyte apoptosis; however, their contribution to the increase of apoptosis in LP B-cells during sepsis is not known. To study this, sepsis was induced by cecal ligation and puncture (CLP) in endotoxin-tolerant C3H/HeJ or FasL-deficient C3H/HeJ-FasL(gld) (FasL(-)) mice and LP lymphocytes were isolated 24 h later. Phenotypic, apoptotic, and functional indexes were assessed. The number of LP B cells decreased markedly in C3H/HeJ mice but not in FasL-deficient animals at 24 h after CLP. This was associated with comparable alteration in apoptosis and Fas antigen expression in the B cells of these mice. Septic LP lymphocytes also showed increased IgA production, which was absent in the FasL-deficient CLP mice. Furthermore, Fas ligand deficiency appeared to improve survival of septic challenge. These data suggest that the increase in B cell apoptosis in septic animals is partially due to a Fas/FasL-mediated process but not endotoxin.     

α-Tocopherol succinate protects mice against radiation-induced gastrointestinal injury

The purpose of this study was to elucidate the role of α-tocopherol succinate (α-TS) in protecting mice from gastrointestinal syndrome induced by total-body irradiation. CD2F1 mice were injected subcutaneously with 400 mg/kg of α-TS and exposed to different doses of (60)Co γ radiation, and 30-day survival was monitored. Jejunum sections were analyzed for crypts and villi, PUMA (p53 upregulated modulator of apoptosis), and apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling - TUNEL). The crypt regeneration in irradiated mice was evaluated by 5-bromo-2-deoxyuridine (BrdU). Bacterial translocation from gut to heart, spleen and liver in α-TS-treated and irradiated mice was evaluated by bacterial culture on sheep blood agar, colistin-nalidixic acid, and xylose-lysine-desoxycholate medium. Our results demonstrate that α-TS enhanced survival in a significant number of mice irradiated with 9.5, 10, 11 and 11.5 Gy (60)Co γ radiation when administered 24 h before radiation exposure. α-TS also protected the intestinal tissue of irradiated mice in terms of crypt and villus number, villus length and mitotic figures. TS treatment decreased the number of TUNEL- and PUMA-positive cells and increased the number of BrdU-positive cells in jejunum compared to vehicle-treated mice. Further, α-TS inhibited gut bacterial translocation to the heart, spleen and liver in irradiated mice. Our data suggest that α-TS protects mice from radiation-induced gastrointestinal damage by inhibiting apoptosis, promoting regeneration of crypt cells, and inhibiting translocation of gut bacteria.     

Mock retroviral infection alters the developmental potential of murine bone marrow stem cells.

Retroviral vectors were used to introduce an activated ras gene into murine pluripotent hemopoietic stem cells. We attempted to reconstitute the hemopoietic system of lethally irradiated mice with isolated spleen colonies obtained in vivo after injection of infected bone marrow cells. Spleen colonies derived from infected bone marrow were inefficient in promoting long-term survival of irradiated hosts. This loss of reconstitutive capacity of spleen colonies was not due to the retroviral infection per se but to the in vitro culture of spleen colony precursors. Incubation for 24 h in the presence of fetal calf serum and interleukin-3 without virus-producing cells was sufficient to abolish completely the reconstitutive capacity of spleen colonies while maintaining both self-renewal and pluripotential capacities of spleen colony precursors. These results show that the in vitro manipulation of stem cells that is included in current protocols for retroviral infection can modify the developmental potential of these cells. This finding clearly indicates that the use of retroviral vectors can introduce a bias in the analysis of hemopoiesis.     

无绿藻对小鼠Th17细胞分化的影响

目的 研究无绿藻是否能够影响小鼠Th17细胞的分化,影响Th17细胞分泌相关的细胞因子.方法 体外培养无绿藻,用尼龙毛柱法分离培养小鼠脾脏T淋巴细胞,按照1∶5的比例将两者在transwell培养板上共培养,培养2h、4h、8h、12 h、24 h、48 h,分别收集T淋巴细胞,留取培养上清液.流式细胞技术检测Th17细胞表型,ELISA法检测培养上清液中IL-17的水平.结果 从共培养2h开始,随着共培养时间的延长Th17细胞所占比例逐渐降低,共培养8h后趋于稳定（P＜0.05）;从共培养2h开始,培养上清液中IL-17的水平逐渐升高,8h后出现下降,24h后趋于稳定（P＜0.05）.结论 在与T细胞共培养的条件下,无绿藻抑制了小鼠Th17细胞的分化,但是在最初2～8h可以促进Th17相关细胞因子IL-17的释放.     

Target cells for the activity of a synthetic adjuvant: muramyl dipeptide.

Muramyl dipeptide (MDP), a synthetic adjuvant, increased the primary response of CBA mice to sheep red blood cells (SRBC). In reconstituted irradiated recipients, cooperation between T and B lymphocytes was required for the expression of adjuvant activity and MDP increased the efficiency of SRBC-educated T cells. The role of T-derived lymphocytes in mediating the MDP adjuvant activity was also demonstrated in irradiated mice and in mice reconstituted with various splenic cellular types of donors which had received SRBC and MDP 24 hr earlier. In our experiments, the macrophage did not seem to be involved, since MDP did not increase the phagocytic capacity of peritoneal exudate cells and MDP- and SRBC-pretreated macrophages had no increased ability to induce an anti-SRBC immune response. These results demonstrate the importance of T lymphocytes as mediators of the adjuvant activity of MDP.     

Oxidative stress by visible light irradiation suppresses immunoglobulin production in mouse spleen lymphocytes

In this study, we attempted to induce the oxidative stress in mouse spleen lymphocytes with visible light irradiation and examined the effects of lipid peroxidation on immunoglobulin (Ig) production. The spleen lymphocytes were isolated from 8-week-old male balb/c mice and irradiated with 300 W visible light. When the cells were cultured for 72 hr, Ig contents in culture supernatants were decreased gradually by irradiation for over 30 min. The cell viability was also lowered by the irradiation. Intracellular phosphatidylcholine hydroperoxide (PCOOH) levels and thiobarbituric acid-reactive substances (TBARS) values in culture supernatants were measured as indices of lipid peroxidation and we found that Ig production by mouse spleen lymphocytes was suppressed accompanied with the progress of peroxidation of intracellular phospholipids. Cell membrane fluidity was also significantly decreased, but the intracellular Ig level was not changed in the irradiated cells. These results suggest that the peroxidation of intracellular lipids is a cause of the suppression of Ig production by mouse spleen lymphocytes via lowering cell viability and suppressing Ig synthesis and secretion.     

Induction of apoptosis by high linear energy transfer radiation: role of p531

The involvement of the tumor suppressor p53 gene in the sensitivity of many cell types towards low linear energy transfer (LET) radiation is now well established. However, little information is available on the relationship between p53 status of tumor cells and their ability to undergo apoptosis following exposure to high-LET radiation. Here we present the results of experiments carried out with the human lymphoblastoid cell line TK6 and its p53 knock-out counterpart NH32. Cells were irradiated at doses ranging from 0.25 to 8 Gy with fast neutrons (65 MeV), carbon ions (95 MeV/nucleon), and X rays (15 MV). For both cell lines, the occurrence of apoptosis, determined by the quantification of hypodiploid particles as well as the activation of several caspases, was compared with their sensitivity towards high-LET radiation. Results indicate that p53 is involved in the response of TK6 cells to fast neutrons and carbon ions, as measured by cell proliferation and occurrence of apoptosis. However, p53-deficient cells are still able to undergo apoptosis following irradiation. This suggests that heavy ions and fast neutrons induce cellular damage that is not under the control of p53. The involvement of executioner caspases in high-LET radiation induced apoptosis was also evaluated by use of specific inhibitors.     

Status of T- and B-lymphocytes in long living CBA--F1 (CBA X C57BL/6) chimeras]

Semiallogeneic chimeras were produced by injecting 3 X 10(7) spleen cells of mice CBA (H--2k, Mlsd) to lethally irradiated mice (CBA X C57BL/6)F1. Two days later recipients were given cyclophosphamide (CP), 2 mg per mouse, to prevent death of graft versus host reaction (GVHR). For 1.5--2 months after the creation of chimerism in 23 of 26 mice under study all cells producing antibodies to SRBC were represented by donor cells of H-2 phenotype; 3 mice were partial chimeras. Spontaneous blast transformation in the cultures of chimera spleen did not exceed the control level, and in the mixed lymphocyte culture chimera cells failed to proliferate on addition of irradiated lymphocytes (CBA X C57BL/6) F1. At the same time chimera gave intensive blast transformation to the irradiated lymphocytes of the third line of mice DBA/2 (H--2d, Mlsa). Among the chimera spleen cells no killers capable of destroying target cells of donor or recipient origin were revealed. Similar results were obtained in vivo: chimera cells gave no positive local GVHR after administration to mice (CBA X C57BL/6) F1. Prolonged chimerism was accompanied by a reactivity of donor T-lymphocytes to the recipient transplantation antigens. A blocking factor was revealed in the blood serum of chimeras. The substitution of donor lymphocytes for the recipient cells begins after 3 to 5 months. At the same period donor T-cell population reconstitutes partially the responsiveness to the recipient antigens and the blocking factor disappears from chimeras blood.     

Evidence for Subpopulation of Mature Lymphocytes within Mouse Thymus

THERE is increasing evidence that thymus cells migrate from the thymus to the peripheral lymphoid tissues where they make up most, if not all, of the thymus-dependent population of lymphocytes^1–3. The term “thymus-derived” is thus appropriately applied to this population. Yet most thymocytes are different from peripheral lymphocytes, both in immunological competence and in surface antigenic characteristics. For example, thymocytes have more theta (θ)⁴ and less H2 antigen⁵ than do peripheral lymphocytes and in TL-positive strains of mice only thymocytes normally express the TL antigen⁶. Recently, Lance et al.⁷ found that injected thymocytes which had migrated to lymph nodes and spleen were progressively less susceptible to anti-TL and more susceptible to anti-H2 serum over the first 24 h. I report here experiments in which thymus cells injected intravenously into irradiated syngeneic mice and harvested as early as 3 h later from the peripheral lymphoid tissues can be shown to have the surface antigenic properties of peripheral thymus-derived lymphocytes rather than thymocytes. A second experiment demonstrates that at least part of the differentiation from thymocyte to thymus-derived lymphocyte seems to occur within the thymus.     

Studies on the maturation of immune responsiveness in the mouse. II. Role of the spleen.

Experiments were designed to investigate the role of the spleen in the development of the murine immune system. By using mice splenectomized within 24 hr of birth, as well as mice with a hereditary, congenital absence of the spleen, the primary immune response to sheep erythrocytes was examined. The immunocompetence of lymph node cells from spleenless or control mice was assessed in vitro, in organ and in cell suspension cultures, and in vivo, by transfer into lethally irradiated syngeneic recipients followed by antigenic stimulation. The immunologic capacities of thymus and bone marrow cells were similarly tested by injection separately or in combination into irradiated syngeneic mice. Lymph node cells from spleenless animals appeared fully competent both in vitro and in transfer experiments. Neither neonatal splenectomy nor congenital absence of the spleen significantly reduced the capacity of bone marrow or thymus cells to participate in the immune response to sheep erythrocytes.     

Collagen-induced arthritis in mice. VI. Synovial cells from collagen arthritic mice activate autologous lymphocytes in vitro

Synovial cells were extracted from normal and collagen-arthritic mice and investigated for lymphocyte-activating properties. In mixed cell culture, irradiated fibroblast-like synovial cells from DBA/1 LacJ arthritic mice stimulated a strong proliferative response in spleen cells from syngeneic normal mice, but not in cells from allogeneic DBA/2. B10.RIII, or BALB/c mice. This novel stimulus occurred in the absence of detectable Class II MHC antigen expression on the fibroblast-like synovial cell surface or increased autologous mixed lymphocyte reactions between DBA/1 LacJ spleen and lymph node cells. Irradiated synovial cells were also unable to present type II collagen to a collagen-specific T cell line and to stimulate proliferation. Addition of interferon-gamma or interleukin-1 failed to induce detectable surface Ia on the synovial fibroblasts or induce the capacity for antigen presentation in these cells.     

Recognition and response to alloantigens in vivo. I. Negative and positive selection of MLR reactivity in murine peripheral blood lymphocytes to major histocompatibility complex and Mls antigens

Specific mixed lymphocyte reaction (MLR) responsiveness to allogeneic major histocompatibility complex (MHC), or minor lymphocyte-stimulating (Mls) determinants, was depleted in the peripheral blood lymphocytes (PBL) obtained from mice 24 to 48 hr after i.v. injection of 5 to 7.5 X 10(7) MHC or Mlsa-incompatible spleen cells, respectively. Results of cell mixture experiments suggest that the generation of suppressor cells was not the explanation for this specific reduction in MLR proliferation occurring with these PBL responder cells. To gain additional insight into parameters involved in the recognition of allodeterminants in vivo, experimental manipulations of the host environment and donor cell inoculum utilized in the negative selection procedure were employed. For example, removal of the spleen in the recipient animal, an anatomic site in which injected allogeneic cells and corresponding host antigen-reactive cells (ARC) are trapped, still permitted the specific depletion in murine PBL of host ARC for donor foreign MHC antigens. This finding may implicate other sites such as the liver where unprimed host alloreactive clones are trapped. In addition, irradiation of allogeneic donor cells significantly reduced their capacity to trap alloreactive T cell clones in vivo, whereas heat treatment of the donor cells completely eliminated this ability, even though the Ia determinants were still expressed, measured by flow cytometry. After the negative selection period, kinetic analysis of proliferation showed that 3, 4, or 5 days after injection of MHC-incompatible allogeneic spleen cells, the PBL of the recipient showed specific hyperresponsiveness to the MHC-haplotype of the donor cells. Interestingly, these primed PBL responder cells had the volume distribution of small resting cells; thoracic duct lymphocytes (TDL), positively selected by adoptive transfer of T cells to irradiated semiallogeneic recipients, are reported to be mainly blast cells. In contrast to the MLR hyperresponsiveness that results from priming with MHC-incompatible splenocytes, PBL, obtained at these later time points from mice primed with Mlsa-incompatible, H-2-compatible splenocytes, showed complete unresponsiveness in MLR to these Mlsa-bearing stimulator cells, as well as some nonspecific reduction in proliferation to MHC-incompatible stimulator cells regardless of their Mls genotype.(ABSTRACT TRUNCATED AT 400 WORDS)