Effects of phenytoin on cell-mediated immunity

Summary The effects of phenytoin on cellular immunity were examined in murine models. Fresh splenocytes were obtained from mice which had received 1 mg/day of phenytoin i.p. for 28 days. The serum concentration of phenytoin in these animals was 10–10 g/ml. The proliferative response of splenocytes to mitogens was assessed by ³H-thymidine incorporation. The cytotoxic activities of cells such as natural killer (NK) cells, cytotoxic T lymphocytes (CTL), and lymphokine-activated killer (LAK) cells were estimated by a 4-h ⁵¹Cr release assay. The ³H-thymidine incorporation of splenocytes was reduced significantly (P<0.01) in phenytoin-treated mice. The NK and CTL activities of splenocytes from phenytoin-treated mice were significantly suppressed. However, the LAK activity of phenytoin-treated mice was equal to that of control mice.     

Potent induction of TNF-α during interaction of immune effectors with oral tumors as a potential mechanism for the loss of NK cell viability and function

The inhibitory role of TNF-α on survival of naïve and IL-2 treated NK cells has been demonstrated in the past. However, its effect on the function of these cells against tumor cells, in particular against oral tumors has not been established. We investigated the significance of secreted TNF-α in death and functional loss of splenocytes and NK cells in ex-vivo cultures with oral tumors. Oral tumors trigger potent secretion of TNF-α by human and murine immune effectors. Absence of TNF-α increases the cytotoxic activity and secretion of IFN-γ by IL-2 treated splenocytes and NK cells in co-cultures with MOK L2D1+/p53?/? oral tumor cells. IL-2 treated splenocytes and NK cells from TNF-α ?/? mice survive and proliferate more when compared to cells from TNF-α +/+ mice. Cell death induced by F. nucleatum, an oral bacteria, in TNF-α ?/? splenocytes are considerably lower than that induced in TNF-α +/+ splenocytes where potent release of TNF-α is reproducibly observed. Addition of exogenous rTNF-α to IL-2 treated splenocytes and NK cells decreased survival and function of splenocytes and NK cells obtained from TNF-α ?/? mice against oral tumors. These findings suggest that potent induction of TNF-α during interaction of immune effectors with oral tumors and/or oral bacteria is an important factor in decreasing the function and survival of cytotoxic immune effectors. Strategies to neutralize TNF-α may be beneficial in the treatment of oral cancers.     

Cytotoxic properties of the splenocytes of C3HA mice following treatment with exogenous RNA

The influence of fractions of exogenous RNA, isolated from spleens of C3HA mice and of rats, both intact (control--cRNA) and immunized with homogenate of normal syngenic, allogenic and xenogenic tissues (immune--immRNA), on the cytotoxic properties of splenocytes of C3HA intact mice was compared in the in vitro cytotoxic experiments. The splenocytes treated with different RNA fractions were used as effector-cells. In vitro cultivated MGXXIIa cells of strain specific C3HA mice hepatoma, and K562 cells of human erythroleukemia, both labeled with 3H-uridine, served as target cells. Thus, it is only the cytoplasmic fraction of immRNA isolated from the spleens of rats immunized with tissue antigens of C3HA mice that induced a more pronounced stimulation of cytotoxic activity of splenocytes.     

Splenocyte functional activity in mice growing the syngeneic transplantable hepatoma 22a

The growth of syngeneic transplantable hepatoma 22a is not accompanied by alteration of functional activity of T-lymphocytes in the spleens of tumor-bearing hosts as evidenced by means of PHA-blasttransformation test. The contents of T-lymphocytes in the spleens of tumor-bearers was non changed. The cytotoxic activity of the splenocytes towards hepatoma 22a cells was increased either in vivo (Winn-test) or in vitro (cytotoxic test) as compared with splenocytes of intact mice. By means of the test of inhibition of peritoneal macrophage adherence, the increased level of sensibilization of splenocytes towards tissue antigens was shown in mice hearing hepatoma 22a.     

Monoclonal antibodies inhibiting the function of a murine cytotoxic T lymphoma

The thymic lymphoma NS8, obtained by infection of murine antigen-primed lymphocytes with the Radiation Leukemia Virus (RadLV) exhibits a cytotoxic function specific for the sarcoma target T2. We have immunized LOU rats with cells from this cytotoxic T lymphoma and fused their splenocytes with cells from the LOU rat myeloma IR983F to obtain hybridomas. Monoclonal antibodies produced by the 1G hybridoma recognize structures on the surface of NS8 cells. Moreover they are able to inhibit the expression of the specific cytotoxicity mediated by NS8 cells. In contrast, the cytolytic activity of a MLC is not affected by these monoclonal antibodies.     

Induction of candidicidal activity in mice by immunization with Candida albicans ribosomes

Abstract Mice immunized with ribosomes from Candida albicans are protected against experimental systemic candidiasis. In this study we investigated the candidacidal activity of spleen cells from immunized animals as measured by ⁵¹Cr release from pre-labelled yeast cells. It was found that the anti-candidal cytotoxic activity of splenocytes from immunized mice was significantly higher than that of spleen cells from non-immunized controls with various effector to target (E:T) ratios, but optimal results were obtained with an E:T ratio of 10:1. The cytotoxic activity of splenocytes as measured by the ⁵¹Cr release assay correlated well with the capacity of the cells to inhibit candidal growth as determined by quantitative plating. This candidacidal activity was not antibody dependent but increased killing was obtained by adding fresh (but not heat inactivated) mouse serum. The enhanced candidicidal activity was inhibited by removal of plastic- or nylon-adherent cells from the cell suspension but not by treatment with anti-Thy 1.2 serum and complement. The data indicate that a candidacidal cell population is induced in the spleens of animals immunized with C. albicans ribosomes.     

Induction of antigen-specific parasiticidal cytotoxic T cell splenocytes by a major membrane protein (P30) of Toxoplasma gondii

Infection with Toxoplasma gondii has become a major cause of morbidity in patients with AIDS. To investigate the mechanisms responsible for immune responses to toxoplasma Ag we used a highly purified membrane protein (P30) of T. gondii to stimulate an in vitro Ag-specific cytotoxic T cell response. P30 immune mouse splenocytes reduced extracellular T. gondii plaque-forming units by more than 50% when incubated at an E/T ratio of 10:1 or greater. By using a [3H]uracil radioisotope release assay, the effect of the immune splenocytes was determined to be a direct parasite lytic mechanism. The immune splenocytes were P30 Ag specific and of the Thy 1.2, Lyt2,3+ (CD4-, CD8+) phenotype, specific for mouse cytotoxic T cells. Opsonization of the parasites with monoclonal P30-reactive mAb did not enhance parasiticidal activity. Culture supernatants obtained during the 2-h cytotoxic assay were not parasiticidal, and anti-asialo-GM1 antibody plus C did not destroy the parasiticidal activity of the P30 responder cells. Accordingly, we have identified an Ag-specific subset of CD4-, CD8+, P30 responder T cells that are directly parasiticidal to extracellular T. gondii, and that exhibit cytotoxicity independent of antibody opsonization, lymphokine secretion, NK cell activity, and, apparently, MHC involvement as well.     

Effect of heated naringenin on immunomodulatory properties and cellular antioxidant activity

Naringenin is one of the most popular flavonoids derived from citrus. It has been reported to be an effective anti-inflammatory compound. Citrus fruit may be used raw, cooked, stewed, or boiled. The present study was conducted to investigate the effect of thermal processes on naringenin in its immunomodulatory and cellular antioxidant activities. The effects of flavonoids on B and T cell proliferation were assessed on splenocytes stimulated or not with mitogens. However, their effects on cytotoxic T lymphocyte (CTL) and natural killer (NK) activities were assessed in splenocytes co-incubated with target cells. The amount of nitric oxide production and the lysosomal enzyme activity were evaluated in vitro on mouse peritoneal macrophages. Cellular antioxidant activity in splenocytes and macrophages was determined by measuring the fluorescence of the dichlorofluorescin (DCF). Our findings revealed that naringenin induces B cell proliferation and enhances NK activity. The highest concentration of native naringenin exhibits a significant proliferation of T cells, induces CTL activity, and inhibits cellular oxidation in macrophages. Conversely, it was observed that when heat-processed, naringenin improves the cellular antioxidant activity in splenocytes, increases the cytotoxic activity of NK cells, and suppresses the cytotoxicity of T cells. However, heat treatment maintains the anti-inflammatory potency of naringenin.     

Immunostimulating effects of muramyl dipeptide, glucosaminyl muramyl dipeptide and their synthetic derivatives in vitro

The effect of muramyldipeptide (MDP), glucosaminylmuramyldipeptide (GMDP) and their six synthetic derivatives on production of tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-2 (IL-2) by murine spleen cells in vitro was studied. MDP induced insignificant TNF production and did not stimulate production of IL-1 by the murine splenocytes within a 24-hour cultivation period whereas in combination with lipopolysaccharide (LPS) it induced significant production of both the cytokins. GMDP induced marked production of TNF (54 per cent cytotoxic index) and IL-1 (stimulation index 8). Addition of LPS in an amount of 10 ng/ml increased production of TNF by the murine splenocytes under the effect of GMDP but had no effect on production of IL-1. Neither MDP nor GMDP even in combination with LPS induced production of IL-2 by splenocytes of mice DVA/2 and C57B1/6 at activation for 24 hours. All the synthetic derivatives of MDP and GMDP except the MDP polymer activated TNF production by the murine spleen cells. GMDP lysine had the highest effect: 67 per cent cytotoxic index. In combination with LPS its cytotoxic index amounted to 87 per cent. The TNF activity was always higher when LPS in an amount of 10 ng/ml was added to the glycopeptides.     

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.     

In vitro elicitation of cytotoxic response against a nonimmunogenic murine tumor by allosensitization

Summary The murine lymphoma (thymoma) PIR-2 of C57BL/6 origin, primarily induced in our laboratory by fractionated X-ray irradiation, has been shown to be nonimmunogenic by its failure to immunize syngeneic mice in vivo or to evoke a cytotoxic response in primary mixed lymphocyte-tumor cell cultures (MLTC) in vitro. We were able, however, to demonstrate the existence of anti-PIR-2 cytotoxic cells among allogeneic-primed C57BL/6 responding lymphocytes using the technique of limiting dilution cultures (LDC). The frequency of anti-PIR-2 cytotoxic cells among C57BL/6 lymphocytes sensitized against BALB/c splenocytes in mixed leukocyte culture (MLC) was 1/20 to 1/40, and the cytotoxic activity of positive LDC wells against PIR-2 reached 60% as determined by a 4-h ⁵¹Cr-release assay. The frequency of anti-PIR-2 cytotoxic cells could be increased two- to 10-fold (up to 1/4) by removing nylon-wool-adherent cells from the primed cell population and/or by enriching the primed lymphoblast population on a Percoll density gradient. Anti-PIR-2 cytotoxic cells were found to be Thy1⁺; Lytl^–2⁺ cells. Clones isolated from the LDC wells manifested strong cytotoxic activity toward PIR-2 cells and the stimulating BALB/c splenocytes but not against other H-2^b tumor lines or C57BL/6 splenocytes. We suggest that the procedure of allostimulation in MLC-LDC is an effective in vitro means of generating highly reactive cytotoxic cells against poorly immunogenic neoplasms.     

Further characterization of suppressor lymphocytes induced by fetal calf serum in murine lymphoid cell cultures: comparison with in vitro-generated cytotoxic lymphocytes.

Nonspecific suppressor cell (SPC) activity has been induced in vitro by preculturing splenocytes from normal mice in the presence of fetal calf serum (FCS) for 3 days or more. In adoptive transfer experiments in vivo, these precultured SPC were shown to reduce the humoral response of mice to SRBC and the cell-mediated cytotoxic (CMC) response to allogeneic tumor cells. In mixing experiments in vitro, using freshly explanted splenocytes, the precultured splenocytes abrogated the generation of specific cytotoxic lymphocytes (CL) in primary mixed lymphocyte-tumor cell cultures (MLTC). By contrast, secondary cytotoxic response was only marginally affected. Supernatants of precultured cells were also inhibitory, although to a lesser degree than whole cells. The induction of suppressive activity was abolished by addition of mitogenic amounts of concanavalin A to the preculturing medium.By the use of cell fractionation techniques it was found that both specific CL and nonspecific SPC lack an Fc receptor, do not adhere to nylon wool, and cannot be separated from each other by density sedimentation on a discontinuous BSA gradient. However, precursors of SPC and CL differed in their susceptibility to cyclophosphamide, hydrocortisone, and irradiation. The data presented does not exclude the possibility that suppressive activity exerted by FCS-induced SPC is mediated through a cytotoxic effect.     

乳杆菌对牛乳β-乳球蛋白致敏小鼠淋巴细胞分泌Th1/Th2型细胞因子和抗体的体外影响

为了分析乳杆菌对致敏小鼠脾淋巴细胞分泌Th1/Th2细胞因子及抗体的体外影响,用牛乳β-乳球蛋白腹腔注射BALB/c小鼠建立过敏症模型,造模成功后,分离致敏小鼠的脾淋巴细胞并与4种活/死乳杆菌(107 CFU/mL)体外共同孵育,ELISA法检测细胞上清液中细胞因子(IL-12、IFN-γ和IL-4)和抗体(总IgE、β-Lg特异性IgE和总IgG)含量。4种活/死乳杆菌均可体外调节致敏小鼠脾淋巴细胞分泌细胞因子和抗体的水平,特别是热致死的发酵乳杆菌和嗜酸乳杆菌可提高淋巴细胞IL-12和IFN-γ的分泌,抑制IL-4的分泌,使其IFN-γ/IL-4比值(代表Th1/Th2细胞平衡)高于活菌,与空白对照组比较差异显著(P<0.05)。同时,这两株热致死菌还可显著下调细胞上清液中总IgE、特异性IgE和总IgG抗体的浓度(P<0.05)。试验结果表明乳杆菌可提高牛乳β-乳球蛋白致敏小鼠脾淋巴细胞的IFN-γ/IL-4比值,进而纠正Th2占优势的Th1/Th2失衡,下调抗体分泌量,且具有菌株特异性。     

Intestinal intraepithelial lymphocytes exert potent protective cytotoxic activity during an acute virus infection

After systemic infection of mice with 104 PFU of lymphocytic choriomeningitis virus (LCMV), infected cells are detected simultaneously in various organs, including spleen and intestinal mucosa. Most notably, virus-infected cells are also present among CD11c+ dendritic cells in the subepithelial area of the small intestinal mucosa. Some of these virus-infected cells are in close spatial association with intestinal intraepithelial lymphocytes (IEL). Therefore, we compared virus-specific cytotoxic activity of CD8 splenocytes with that of IEL subsets. While ex vivo isolated TCRalphabeta+CD8alphaalpha+ IEL exert only minimal virus-specific cytotoxicity, maximum specific killing mediated by TCRalphabeta+CD8alphabeta+ IEL on day 8 postinfection exceeds maximum cytotoxic activity observed with CD8 splenocytes when assessed in vitro. Maximum cytotoxic activity of IEL is preceded by peak perforin and granzyme B mRNA expression in IEL around day 6 postinfection, suggesting a recent activation in situ. The antivirus cytotoxicity of in vivo primed IEL is further demonstrated by the protection from virus production in the spleen of mice infected with LCMV 10 h before adoptive cell transfer. These data indicate a potent priming of LCMV-specific IEL in situ after systemic LCMV infection and suggest that cytotoxic IEL markedly contribute to the elimination of virus-infected cells in the intestinal mucosa.     

Induction of cytotoxicity from fresh splenocytes after in vivo administration of cyclophosphamide

Summary Cyclophosphamide, combined with lymphokine-activated killer (LAK) cells and recombinant interleukin-2 (rIL-2), is known to mediate regression of tumors, but the effects of cyclophosphamide on the subsequent generation of LAK cells are unclear. It was the aim of the experiments in this paper to determine whether fresh splenocytes cultured with rIL-2 would maintain or regain their cytotoxicity in vitro after being exposed to the cytotoxic agent cyclophosphamide in vivo. Functional monitoring of splenocytes after in vitro incubation with rIL-2 was performed at various times through chromium-release assays, thymidine assays and cell-cycle analysis. Chromium-release assays determined that the cytotoxicity of cultured splenocytes returned to normal after 12 days of in vitro culture with rIL-2. The thymidine assays indicated a normal rate of uptake of thymidine after 7 days in culture, while the cell cycle was still abnormal by day 12 of culture. The growth and expansion of rIL-2-activated splenocytes after different times of in vitro culture indicated a return to normal compared to control animals after 7 days of continuous in vitro exposure to rIL-2. It is concluded that murine splenocytes can demonstrate cytotoxicity after exposure to cyclophosphamide, through prolonged continuous in vitro culture with rIL-2. Since cyclophosphamide did not jeopardize the production of splenocyte cytotoxic effectors generated with rIL-2, it appears to be a strong contender for use in chemoimmunotherapy protocols.Supported in part by grants from The Alberta Heritage Foundation for Medical Research and the National Cancer Institute of Canada     

An extract of Ulmus macrocarpa improves cellular immunity in immuno-suppressed models

An extract of Ulmus macrocarpa Hance, commonly known as the large-fruited elm, has been prescribed as a traditional medicine. In this study, we aimed to investigate the cellular immune effects of U. macrocarpa stem cortex extracts on cyclophosphamide (CY)-treated splenocytes and mice. A methanol extract showed an improved survival rate of splenocytes after 72?h. The extract was successively partitioned with dichloromethane, ethyl acetate, n-butanol, and water; and the fractions so obtained were also examined for their proliferative activity. Among them, the water fraction of U. macrocarpa showed the highest proliferation of splenocytes and was used throughout the present study. We tested the survival rate of splenocytes through dose-dependent treatment of CY and the suppression of the survival effect by CY was recovered by treatment with the water extract of U. macrocarpa. To determine the mechanism involved, we examined the expression of B-cell lymphoma-extra large (Bcl-xL) anti-apoptotic protein. CY decreased Bcl-xL protein levels in resting splenocyte cultures, whereas splenocytes were exposed to water extracts of U. macrocarpa in the presence of CY; however, elevations in Bcl-xL were observed at 96?h. Mice splenocytes treated with water extract of U. macrocarpa for cellular immunity showed an increase in the activity of the mixed lymphocyte reaction (MLR), cytotoxic T lymphocytes (CTLs), and natural killer (NK) cells. In addition, mice receiving a water extract of U. macrocarpa recovered the CTL, NK, and MLR activities suppressed by CY administration. Consequently, U. macrocarpa improves the cell-mediated immune response and provides an insight on cell-based tonic materials.     

In vivo and in vitro macrophage activation induced by IFN gamma spontaneously released by spleen cells from tumor bearing mice.

Peritoneal macrophages obtained from Lewis Lung carcinoma (3LL) tumor bearing mice release high amounts of soluble factors such as C3,H2O2 and lysosomal enzymes but fail to exert cytotoxic activity on tumor cells. In the present work we show that they acquire this property and become fully activated after in vitro incubation with supernatants derived from cultures of splenocytes from tumor bearing syngeneic mice. The presence of IFN gamma in the above supernatants was detected by immunoblotting analysis and by bioassay. The role played by IFN gamma in macrophage activation was investigated.     

Induction of cell-mediated cytotoxic immunity to sperm-specific lactate dehydrogenase-C4 in SJL/J female mice

SJL/J female mice were tested for a cell-mediated cytotoxic response to sperm-specific lactate dehydrogenase C4 (LDH-C4). We demonstrated this response with a 51chromium release assay. Mouse LDH-C4 was coupled to EL-4 tumor cells. These cells were then labeled with 51Cr and mixed with splenocytes from LDH-C4-immune and -nonimmune mice. Specific lysis of the tumor cells by splenocytes from LDH-C4-immune mice was detected at 7 days and 14 days following a single footpad immunization. Since LDH-C4 is present on the surface of sperm, these results support the suggestion that cytotoxic removal of sperm from the female reproductive tract is one of the mechanisms whereby fertility is reduced following immunization with this enzyme.     

Production of cytokines by peritoneal macrophages and splenocytes after exposures of mice to low doses of X-rays

We have shown previously that irradiations of mice with 0.1 or 0.2 Gy of X-rays stimulate anti-tumour cytotoxic activities of peritoneal macrophages and splenocytes enriched for NK lymphocytes and suppress the development of pulmonary tumour colonies. The up-regulated cytotoxicities were related to the production of nitric oxide by macrophages, and perforin and Fas ligand by the splenocytes, but specific blockade of these pathways did not totally suppress the effector cell-mediated cytolysis of the tumour target. Hence, other factors such as cytotoxic/cytostatic cytokines might have been produced by the effector cells. To test this possibility peritoneal macrophages and splenocytes were isolated from BALB/c mice which had been either once or tentimes whole body-irradiated with the total doses of 0.1 and 0.2 Gy of X-rays and assayed for the levels of IL-1beta, IL-2, IL-12, IFN-gamma and TNF-alpha in the incubation medium using the respective ELISA kits. The results demonstrate that both single and multiple exposures to the two low doses of X-rays significantly stimulate secretion of IL-1beta, TNF-alpha and IL-12 by macrophages and IL-2 and IFN-gamma by splenocytes, but the kinetics and magnitude of the induced changes in the production of these cytokines differ between the two irradiation protocols.     

Dimeric sesquiterpene thioalkaloids with potent immunosuppressive activity from the rhizome of Nuphar pumilum: structural requirements of nuphar alkaloids for immunosuppressive activity

Potent immunosuppressive dimeric sesquiterpene thioalkaloids, 6-hydroxythiobinupharidine, 6,6'-dihydroxythiobinupharidine, 6-hydroxythionuphlutine B and 6'-hydroxythionuphlutine B, were isolated from the rhizome of Nuphar pumilum together with five inactive quinolizidine alkaloids, neothiobinupharidine, nupharidine, deoxynupharidine, 7-epideoxynupharidine and nupharolutine. These dimeric sesquiterpene thioalkaloids were found to significantly inhibit anti-sheep erythrocyte plaque forming cell formation in mouse splenocytes at 1 microM. At this concentration. 6-hydroxythiobinupharidine, 6-hydroxythionuphlutine B and 6'-hydroxythionuphlutine B did not show cytotoxic effects to mouse splenocytes, and 6,6'-dihydroxythiobinupharidine also showed only minor or minimal cytotoxicity. By comparison of the inhibitory activity of several Nuphar alkaloids on anti-sheep erythrocyte plaque forming cell formation, some structural requirements of Nuphar alkaloids for immunosuppressive activity were obtained. Namely, the 6- or 6'-hydroxyl group at the quinolizidine ring of dimeric sesquiterpene thioalkaloids is essential for the immunosuppressive effect. The number of hydroxyl groups appears to be related to the cytotoxicity, and the influence on splenocytes is greater with increasing numbers of hydroxyl groups.