Radiation and primary immune response to lipopolysaccharide: lymphocyte distribution and function.

INTRODUCTION: Lipopolysaccharide (LPS) is a major cause of septic shock and death due to infection with Gram-negative bacteria. The purpose of this study was to quantify the effects of whole-body irradiation on lymphocyte populations during response to challenge with LPS. MATERIALS AND METHODS: C57BL/6 mice (n = 10/group) were irradiated whole-body with 3 gray (Gy) gamma-rays in a single fraction at 0.8 Gy/min. LPS (E. coli serotype 0111:B4) at 1 mg/kg was injected intraperitoneally 10 days later and mice were euthanized at 60 min and days 1, 7, and 14 post-inoculation for analyses. RESULTS: Significant interactions between radiation and LPS were noted in circulating and splenic lymphocyte subpopulations, including T-, B-, and NK-cells, particularly at the early time points. There were significant interactions on circulating, but not splenic, CD62L+ T-cell populations. However, there were no interactions on CD62L+ B-cells. Finally, there were significant interactions in both early and late blastogenic responses. CONCLUSION: The data support that response to infection with Gram-negative bacteria may be significantly compromised by exposure to ionizing radiation.

Acute effects of iron-particle radiation on immunity. Part I: Population distributions.

Health risks due to exposure to high-linear energy transfer (LET) charged particles remain unclear. The major goal of this study was to confirm and further characterize the acute effects of high-LET radiation ((56)Fe(26)) on erythrocyte, thrombocyte and leukocyte populations in three body compartments after total-body exposure. Adult female C57BL/6 mice were irradiated with total doses of 0, 0.5, 2 and 3 Gy and killed humanely 4 days later. Body and organ masses were determined and blood, spleen and bone marrow leukocytes were evaluated using a hematology analyzer and flow cytometry. Spleen and thymus (but not body, liver and lung) masses were significantly decreased in a dose-dependent manner. In general, red blood cell (RBC) counts and most other RBC parameters were depressed with increasing dose (P < 0.05); the major exception was an increase in cell size at 0.5 Gy. Platelet numbers and volume, total white blood cell counts, and all three major types of leukocytes also decreased (P < 0.05). Lymphocyte populations in blood and spleen exhibited variable degrees of susceptibility to (56)Fe-particle radiation (B > T > NK and T cytotoxic > T helper cells). In the bone marrow, leukocytes with granulocytic, lymphocytic ("dim" and "bright"), and monocytic characteristics exhibited proportional variations at the higher radiation doses in the expression of CD34 and/or Ly-6A/E. The data are discussed in relation to our previous investigations with iron ions, other forms of radiation, and space flight in this same animal model.

Acute effects of iron-particle radiation on immunity. Part II: Leukocyte activation, cytokines and adhesion.

The effects of high-linear energy transfer (LET) radiation on immune function have not been clearly established. The major goal of this study was to evaluate leukocyte responses after whole-body exposure to high-LET radiation. C57BL/6 mice were exposed to 0, 0.5, 2 and 3 Gy (56)Fe(26+) particles (1055 MeV/nucleon, 148.2 keV/microm) and killed humanely 4 days after exposure. Spontaneous synthesis of DNA in blood and spleen cells was increased significantly in groups receiving either 2 or 3 Gy (P < 0.001). In contrast, a significant depression in the response of T lymphocytes to phytohemagglutinin (PHA) and concanavalin A (ConA) was noted (P < 0.005); the response to lipopolysaccharide (LPS), a B-cell mitogen, was similar among groups. A cytometric bead array assay revealed that the level of tumor necrosis factor alpha (Tnfa) secreted by splenocytes increased significantly with increasing (56)Fe-particle dose (P < 0.05); interferon gamma, interleukin2 (Il2), Il4 and Il5 were unaffected. Flow cytometry analysis showed that 2 and 3 Gy markedly reduced splenic mononuclear cells expressing the activation markers CD25 and CD71, both with and without the T-cell marker CD3 (P < 0.05); proportions also varied significantly. Similar patterns were noted in mononuclear and granular cells with adhesion markers CD11b and, to a lesser extent, CD54 (P < 0.05). The results show that a single, acute exposure to high-LET radiation induced changes that can profoundly alter leukocyte functions. The implications of the data are discussed in relation to low-LET radiation, altered gravity, and space flight.

Radiation enhances the anti-tumor effects of vaccinia-p53 gene therapy in glioma.

The overall goal of this study was to analyze the effect and mechanism of radiation in combination with vaccinia viruses (VV) carrying the p53 gene against glioma. Comparison of two alternative treatments of cultured C6 (p53(+)) and 9L (p53(-)) rat glioma cells showed significantly reduced survival for both cell lines, especially 9L, when radiation was applied prior to virus versus radiation alone. High p53 protein expression mediated by VV-TK-p53 was measured in infected cells. Single modality treatment of C6 cells with psoralen and UV (PUV)-inactivated VV-TK-p53 (PUV-VV-TK-53) or radiation significantly decreased survival compared with PUV-inactivated L-15 (PUV-L-15) control virus. However, no difference was observed between radiation and combination treatments of C6 cells. In contrast, radiation followed by PUV-VV-TK-53 resulted in dramatic reduction of 9L cell viability, compared to single modality treatment. Flow cytometry analysis of Annexin-V-stained 9L cells showed that radiation and PUV-VV-TK-53 caused a significant decrease in live cells (17.2%) as compared to other treatments and control (61.6-98.3%). Apoptosis was observed in 37.2% of cells, while the range was 0.7-7.8% in other treatment groups; maximal p53 level was measured on day 7 post-infection. In athymic mice bearing C6 tumors, VV-TK-53 plus radiation in both single and multiple therapies resulted in significantly smaller tumors by day 30 compared to the agents given only once. Immunohistochemical analysis of tumor sections demonstrated p53 protein expression over 20 days after VV-TK-53 treatment. Analysis of blood and spleen cells of mice given multiple combination treatments showed significant splenomegaly, leukocytosis, and increased DNA synthesis and response to mitogen. Multiple combination treatments were also associated with significantly elevated natural killer and B cells in the spleen. There were no overt toxicities, although depression in red blood cell and thrombocyte parameters was noted. Collectively, the data demonstrate that radiation significantly improves the efficacy of VV-mediated tumor suppressor p53 therapy and may be a promising strategy for glioma treatment. Furthermore, the results support the conclusion that the mechanisms underlying the enhanced anti-tumor effect of combination treatment include apoptosis/necrosis and upregulation of innate immune defenses.

Evaluation of TNF-alpha/Bax gene therapy and radiation against C6 glioma xenografts.

Successful therapy of high-grade tumors of the brain is likely to require a combination of new therapeutic approaches. The major goal of the present study was to construct a plasmid-based bax gene vector (pGL1-Bax) and evaluate its expression in vitro and in vivo using athymic mice with subcutaneously growing C6 glioma. Preliminary experiments of efficacy and safety were also performed using pGL1-Bax alone and in combination with previously constructed pGL1-TNF-alpha, as well as with radiation. pGL1-Bax was expressed by C6 cells and was correlated with apoptosis, indicating that the construct and the bax protein were functional. Although intratumoral injections of pGL1-Bax alone, up to total doses of 450 micro g, did not significantly affect tumor growth, consistently smaller tumors were obtained when pGL1-TNF-alpha plus pGL1-Bax were injected 16-18 hr prior to tumor irradiation. Furthermore, in mice with two tumors, one treated and one untreated, progression of the untreated tumor was delayed in the animals receiving all three modalities. No prohibitive toxicities were noted, based on mouse body weights and in vitro assays of blood and spleen. Significant increases in spleen mass, total leukocyte counts, percentage of granulocytes, spontaneous blastogenesis, and CD71-expressing B cells were primarily associated with tumor presence and not treatment type. Overall, the results are promising and suggest that TNF-alpha/Bax gene therapy may be beneficial against highly malignant tumors of the brain. To our knowledge, this is the first report of bax gene therapy used together with radiation in an in vivo glioma model.

Modulation of innate immunological factors by STEALTH liposome-encapsulated tumor necrosis factor-alpha in a colon tumor xenograft model.

BACKGROUND: Previous studies have shown that tumor necrosis factor-alpha (TNF-alpha) encapsulated in sterically-stabilized PEGylated STEALTH liposomes (SL) can better and more safely augment the efficacy of other treatment modalities than free TNF-alpha. The aim of this study was to examine the effects of SL-TNF-alpha in the LS174T human colon tumor xenograft model and to correlate its administration with alterations in innate immune system parameters. MATERIALS AND METHODS: Nude mice (n = 128) were injected subcutaneously with LSI 74T cells and treated intravenously with SL-TNF-alpha SL-placebo, or free recombinant human TNF-alpha; the animals were euthanized at 6, 18, 36 and 96 hours after injection. RESULTS: Significant increases in leukocyte, granulocyte, monocyte and NK cell numbers were observed early (6 hours) in the blood from both SL-TNF-alpha and free TNF-alpha treated mice compared to the control group. In contrast, during the 18- to 36-hours interval, SL-TNF-alpha induced significantly higher (p<0.05) leukoctyte, T cell, and NK cell numbers, basal leukocyte proliferation, and CD25+ activation marker expression; the modulation occurred primarily in the spleen. CONCLUSION: These data indicate that both SL-TNF-alpha and free TNF-alpha can induce dramatic up-regulation in leukocyte populations early after injection. However, the up-regulation produced by SL-TNF-alpha was more prolonged and pronounced than that of TNF-alpha and had better correlation with cell activation. These findings suggest that sustained leukocyte recruitment and/or activation may be an important factor in the greater than additive or synergistic antitumor effects observed when SL-TNF-alpha is used in combination with other cancer therapies.

Lymphocyte activation with localized pGL1-TNF-alpha gene therapy in a glioma model.

The major goal of this study was to evaluate the effects of tumor necrosis factor-alpha (TNF-alpha), delivered as pGL1-TNF-alpha, on hematological variables, as well as C6 tumor growth in athymic mice treated with and without radiation. pGL1-TNF-alpha was administered intratumorally at low to high doses (15, 150 and 450 microg) in all three phases of this study. In phase A, pGL1-TNF-alpha expression within tumors was dose dependent and transient, with highest levels seen at 18 h after injection, whereas no TNF-alpha protein was detected in plasma. Low erythrocyte counts, hemoglobin, and hematocrit were associated with tumor presence, but the reduction in these variables was most striking in the group receiving 450 microg of pGL1-TNF-alpha, the group that also exhibited thrombocytopenia at 72 h. In phase B, treatment with pGL1-TNF-alpha at 15 or 150 microg resulted in the greatest degree of splenomegaly, increased spontaneous blastogenesis by splenocytes, and high leukocyte and lymphocyte numbers in the spleen. In these same two groups, flow cytometry analyses of spleen cells showed that high levels of natural killer (panNK+) cells, B (CD19+) lymphocytes, and cells expressing the CD71 and CD25 activation markers were present (p < 0.05). An enhancing effect was also noted in some of the measurements with parental plasmid p WS4 and tumor presence. In phase C, the slowest tumor progression was observed in the groups receiving 15 and 150 microg pGL1-TNF-alpha together with radiation; tumor volumes were 51 and 43% smaller, respectively, than for PBS-injected controls by the end of the study. Collectively, these results show that localized treatment with pGL1-TNF-alpha is hematologically nontoxic at low doses and support the premise that activation of lymphocytes may contribute to the antitumor effects of radiation against a highly aggressive brain tumor.

Dose and dose rate effects of whole-body proton irradiation on leukocyte populations and lymphoid organs: part I.

The goal of part I of this study was to evaluate the effects of whole-body proton irradiation on lymphoid organs and specific leukocyte populations. C57BL/6 mice were exposed to the entry region of the proton Bragg curve to total doses of 0.5 gray (Gy), 1.5 Gy, and 3.0 Gy, each delivered at a low dose rate (LDR) of 1 cGy/min and high dose rate (HDR) of 80 cGy/min. Non-irradiated and 3 Gy HDR gamma-irradiated groups were included as controls. At 4 days post-irradiation, highly significant radiation dose-dependent reductions were observed in the mass of both lymphoid organs and the numbers of leukocytes and T (CD3(+)), T helper (CD3(+)/CD4(+)), T cytotoxic (CD3(+)/CD8(+)), and B (CD19(+)) cells in both blood and spleen. A less pronounced dose effect was noted for natural killer (NK1.1(+) NK) cells in spleen. Monocyte, but not granulocyte, counts in blood were highly dose-dependent. The numbers for each population generally tended to be lower with HDR than with LDR radiation; a significant dose rate effect was found in the percentages of T and B cells, monocytes, and granulocytes and in CD4(+):CD8(+) ratios. These data indicate that mononuclear cell response to the entry region of the proton Bragg curve is highly dependent upon the total dose and that dose rate effects are evident with some cell types. Results from gamma- and proton-irradiated groups (both at 3 Gy HDR) were similar, although proton-irradiation gave consistently lower values in some measurements.