Dissociation between interferon production induced by phytohemagglutinin and concanavalin A in spleen cell cultures of nude mice

Spleen cells of homozygous nude mice did not react with lymphoproliferation to both phytohemagglutinin (PHA) and concanavalin A (Con A), and there was no detectable interferon production in response to Con A. However, PHA-induced interferon production was equal in spleen cells of nu/nu mice and nu/+ mice. One possible explanation for these findings is that a subset of T cells responds to PHA with interferon production different from that responding to Con A with interferon production.     

Suppression of interferon production in mouse spleen cells by cytochalasin D.

Cytochalasin D is thought to impair microfilament function. The present study has investigated its effects on four different systems in which interferon is formed, namely (1) mouse fibroblasts induced with virus (2) mouse spleen cells induced with virus, or (3) with endotoxin or (4) by allogeneic stimulation. Cytochalasin D did not suppress formation of interferon by fibroblasts (L cells) or spleen cells stimulated with either HVJ or NDV. However it did suppress production of interferon by spleen cells in response to endotoxin or an allogeneic stimulation; here its action was apparently not on the secretion of interferon, but on some earlier event. It also suppressed the production of interferon by mouse spleen cells induced with HVJ if this had been u.v. irradiated for more than 15 min: this suggests that cytochalasin D sensitive structures do play some role in interferon production by mouse spleen cells when stimulated with HVJ, as well as when they are stimulated with endotoxin or an allogeneic stimulus.     

Nitric oxide induction by pertussis toxin in mouse spleen cells via gamma interferon.

We examined the major pathogenic substances of Bordetella pertussis for the ability to induce nitric oxide, and important biological function of macrophages, via gamma interferon in spleen cells. B. pertussis, which produces a variety of pathogenic substances, including pertussis toxin and filamentous hemagglutinin, causes a severe respiratory disease. Nitric oxide was detected in the culture fluid of spleen cells stimulated with pertussis toxin or its B oligomer but not in the culture fluid of spleen cells stimulated with the A protomer of pertussis toxin or with filamentous hemagglutinin. Incubation of the peritoneal exudate macrophages with pertussis toxin, B oligomer, A protomer, or filamentous hemagglutinin induced little nitric oxide, whereas incubation with gamma interferon induced a significant amount of nitric oxide. The induction of nitric oxide in spleen cells stimulated with pertussis toxin was completely inhibited by anti-gamma interferon antibody. The treatment of spleen cells with anti-Thy-1.2 antibody plus complement followed by stimulation with pertussis toxin decreased the secretion of gamma interferon and nitric oxide. These results suggest that gamma interferon from T lymphocytes stimulated with pertussis toxin induces nitric oxide.     

The mechanism of interferon induction in mouse spleen cells stimulated with HVJ.

This study showed that functional viral RNA and the penetration of virus into the cell are needed for interferon induction in L cells, while simple contact of the viral glycoprotein with the cell surface appears to be sufficient for interferon induction by the same HVJ in mouse spleen cells. Thirty minutes of uv irradiation resulted in complete loss of the interferon-inducing ability of HVJ in mouse L cells. In contrast to this result, HVJ irradiated for 2 hr could induce interferon in mouse spleen cells as efficiently as untreated HVJ. These findings showed that the actual inducer of interferon in mouse spleen cells was not viral nucleic acid, but some other viral component. When HVJ was treated with potassium periodate at 37° for 1 hr, infectivity for eggs and the hemolytic and neuraminidase activities of the virus were not detectable, but a considerable portion of its hemagglutinating activity was retained. The binding to erythrocytes of this inactivated HVJ, which showed no interferon-inducing ability in both L and mouse spleen cells, was restored in mouse spleen cells but not in L cells. The results indicated that hemolytic and neuraminidase activities are not essential for interferon induction in mouse spleen cells and that hemagglutinating activity might be closely related to interferon induction in the cells, although the presence of hemagglutinating activity alone is not sufficient for interferon induction in the cells. It seems that structural integrity of hemagglutinin on the erythrocyte surface may be important for interferon induction. HeLa cell-grown HVJ, which is characterized by its inability to penetrate into tissue culture cells, was found to stimulate interferon production in mouse spleen cells but not in L cells. This suggests that the process of virus penetration may be essential for induction of interferon in L cells. Interferon was produced in mouse spleen cells incubated with membranous particles with HVJ glycoproteins, but interferon activity could not be detected in the culture fluids of L cells. Aggregation of the glycoproteins by an antibody enhanced its interferon-inducing ability in mouse spleen cells. These results showed that the actual inducer of interferon in mouse spleen cells is not viral nucleic acid, but viral glycoproteins of HVJ, and that the size of its membranous structure is related to interferon inducibility. The mechanism of interferon induction by influenza virus in mouse spleen cells is similar to that of interferon induction by HVJ.     

Role of T and adherent cells in in vitro response of nude mouse spleen cells to bacterial lipopolysaccharide.

Thy 1.2-positive cells and adherent cells were depleted from nude mouse spleen cell suspensions by treatment with anti-Thy 1.2 antisera plus complement and by passage through Sephadex G-10 column, respectively. The results of 3H-TdR uptake and appearance of IgM containing cells induced in vitro by lipopolysaccharide (LPS) were similar in both untreated and depleted spleen cells. Our findings confirm T and adherent cell independence of LPS response in nude mice.     

Component(s) of Sendai virus that can induce interferon in mouse spleen cells.

To identify the active component of Sendai virus that induces interferon in mouse spleen cells, infectious and noninfectious viruses, envelope particles derived from them, and isolated hemagglutinin-neuraminidase (HN) glycoproteins were examined for interferon induction. The interaction between membranous structures containing Sendai virus HN glycoprotein and the receptors on the cell surface was shown to be sufficient for interferon induction in mouse spleen cells, suggesting that the actual inducer of interferon in mouse spleen cells is the HN glycoprotein of Sendai virus. When mouse spleen cells were stimulated in vitro with Sendai virus grown in eggs or LLC-MK2 cells or with membranous structures containing glycoproteins obtained from these viruses, interferon could be detected in the culture fluid. Furthermore, isolated HN glycoprotein per se could induce interferon in the cells. A linear correlation was found between the titer of interferon induced and the hemagglutinating activity of the membranous structure containing the HN glycoprotein. It was concluded from these findings that HN glycoprotein was the active component of Sendai virus responsible for interferon induction in mouse spleen cells and that viral RNA and F glycoprotein were not required. The results also showed that the interaction between HN glycoprotein and receptors on the cell surface triggered production of type I interferon (IFN-alpha and IFN-beta). Although when Sendai virus was incubated at 56 degrees C for 5 min it lost its hemolytic and hemagglutinating activities, it induced a considerable amount of interferon in the culture fluid of mouse spleen cells. The interferon-inducing ability of heat-inactivated virus could be absorbed with mouse spleen cells but not with sheep erythrocytes or mouse erythrocytes, indicating that the inactivated virus retained ability to bind to mouse lymphoid cells.     

Formation of immune (gamma) interferon induced by staphylococcal enterotoxin and phytohemagglutinin

Some conditions of production of human and mouse immune interferon induced by phytohemagglutinin and a national preparation of staphylococcal enterotoxin A (SEA) were studied. The latter was shown to be suitable for use as an inducer of this type of interferon. Production of mouse immune interferon could be increased by using a mixture of spleen and peritoneal exudate cells. Interferon production induced by SEA was also higher in spleen cells of mice immunized with BCG.     

Molecular species of interferon induced in mouse L cells by Newcastle disease virus and polyriboinosinic-polyribocytidylic acid.

Interferons were stimulated in mouse L cells by Newcastle disease virus (NDV) or by polyriboinosinic-polyribocytidylic acid poly(rI).poly(rC). These were fractionated by sequential affinity chromatography on bovine plasma albumin (BPA)-Sepharose and on omega-carboxypentyl (CH)-Sepharose. Based on their interaction with CH-Sepharose, interferon induced by NDV was resolved into three major bands of activity (L/NDV-1,2,3) and poly(rI).poly(rC)-interferon into two (L/rI:rC-1,2). These interferon components were purified to a specific activity of 3 X 10(7) to 4 X 10(7) units/mg protein by antibody affinity chromatography and examined by electrophoresis in SDS-polyacrylamide gels. A total of five molecular species was thus identified for NDV-induced interferon and three for poly(rI).poly(rC) induced interferon, as summarized in Table 1. We conclude from our observations that mouse interferons can be produced by L cells in multiple forms with specific physiochemical properties and in proportions determined by the type of agent employed for induction.     

Interferon production and natural killer activity induced by staphylococcal enterotoxin in mouse spleen cells

After a single intraperitoneal inoculation of C57BL/6 mice with enterotoxin A of Staphylococcus aureus (SEA) the activity of natural killers (NK) increases and phase change of interferon production by spleen cells upon reinduction in vitro occurs. Multiple daily inoculations of mice with SEA maintain the activity of splenic NK at a similar high level. With an adequate control (multiple administration of the medium) NK activity was maintained at the same high level. Interferon production by spleen cells of mice which were multiply inoculated with the medium upon reinduction in vitro was the same as in the control animals, whereas after multiple inoculations of mice with SEA, spleen cells in vitro produced lower amounts of interferon.     

Enhanced gamma interferon responses of mouse spleen cells following immunotherapy for tuberculosis relapse

Gamma interferon responses of spleen cells in mice were examined during postchemotherapy relapse of intraperitoneally induced latent tuberculous infection. The mycobacterial extract RUTI, which prevented the relapse, significantly enhanced the immune responses to secreted and structural recombinant mycobacterial antigens, suggesting that RUTI-mediated protection was mediated by activated T cells.     

转染FasL基因树突状细胞诱导异基因小鼠脾脏T细胞凋亡的研究

目的 制备稳定表达FasL蛋白的小鼠骨髓源树突状细胞(dendritic cell,DC)并探讨其诱导异基因小鼠脾脏T细胞凋亡的机理.方法 采用培养基选择法体外培养小鼠骨髓源DC,脂质体法转染FasL基因至小鼠成熟DC,实时定量PCR检测转染前后FasL mRNA的表达,流式细胞仪和免疫蛋白印迹检测转染前后FasL蛋白的表达.异系小鼠静脉分别输注未转染DC、转染空质粒DC和转染FasL的DC,7 d后TdT介导的原位末端标记法(TUNEL)和流式细胞仪检测脾脏中T淋巴细胞凋亡.结果 体外培养可获得成熟的小鼠骨髓源DC,转染FasL基因的DC较未转染DC FasL mRNA和FasL蛋白表达明显升高.对脾脏中T淋巴细胞凋亡的检测发现,转染FasL的DC组凋亡指数(11.67±1.53)明显高于未转染DC组(2.67±0.58)和转染空质粒组(3.33±0.58),P＜0.01.结论 培养基选择法可收获大量骨髓源DC,脂质体转染FasL基因至小鼠骨髓源DC,可以使DC高表达FasL蛋白.转染FasL的DC输注能明显诱导异系小鼠脾脏T淋巴细胞凋亡.     

Abrogation of tumor induced Ly49 expression on mouse spleen cells by mitomycin C

Mouse spleen cells were activated with IL2 for 4 days in the presence or absence of paraformaldehyde fixed YAC (PFY) tumor cells (spleen cell: PFY ratio 100:1). Fresh spleen cells had poor expression of Ly49A but the expression was significantly upregulated by IL2 activation. Addition of PFYs resulted in a further boosting of Ly49A expression. Besides Ly49A, the Ly49C receptors were also upregulated by PFYs. Upregulated Ly49 expression was not restricted to NK cells (NK1.1 positive cells) but was also seen on T cells (TCRbeta positive cells). Time kinetics studies indicated that maximum upregulation of Ly49 in response to PFY cells occurred on day 3 and 4 and the expression declined thereafter. Ly49 expression in response to PFYs was completely blocked if spleen cells were pre-treated with Mitomycin C, an inhibitor of DNA synthesis. These results show that the addition of a small number of paraformaldehyde fixed YAC cells to spleen cell cultures undergoing IL2 activation, resulted in a significant upregulation of Ly49 receptors and this process was dependent upon cell proliferative activity.     

Recombinant interleukin 2 allows the differentiation of Thy 1.2+ LAK cells from nude mouse spleen cells

Culture of nude mouse spleen cells with recombinant human interleukin 2 (r-IL 2) resulted in the proliferation and generation of lymphokine-activated killer (LAK) cells which could lyse a variety of tumor cells. Flow cytometry study indicated that nude mouse spleen cells contained almost no Thy 1.2+ cells at the initial times of the culture, whereas LAK cells obtained from nude mouse spleen cells by culture with r-IL 2 (nude-LAK cells) expressed high intensity of Thy 1.2 antigen and lymphokine-activated cell-associated (LAA) antigen. The cytotoxic activity of nude-LAK cells was greatly reduced by treatment with anti-Thy 1.2 antibody plus complement but not with anti-Ly 1.2 or Ly 2.2 antibody plus complement treatment. Moreover, nude-LAK cells were resistant to the treatment with anti-asialo GM1 antibody plus complement, in contrast to resident nude natural killer (NK) cells. These data strongly suggested that r-IL 2 allowed the nude mouse spleen cells to differentiate into Thy 1.2+, Ly 1-,2-, asialo GM1- LAK cells which were distinct from Thy 1.2+, Ly 2+, asialo GM1- LAK cells induced from normal mouse spleen cells.     

Immune response induced by spike protein from transmissible gastroenteritis coronavirus expressed in mouse mammary cells

The present study is undertaken to investigate the immune response that was induced by the recombinant spike (S) protein from swine-transmissible gastroenteritis virus (TGEV) expressed in mouse mammary cells. A mammary-specific expression vector pEBS containing the full-length cDNA of S gene was constructed and expressed in the mouse mammary cells (EMT6). The recombinant S protein from culture supernatant of transgenic EMT6 was harvested and immunized BALB/c mice. The results demonstrated recombinant S protein was expressed at high levels in mammary cells by Western blotting and enzyme-linked immunosorbent assay (ELISA) detection. The antibody titer in BALB/c mice following immunization with recombinant S protein was detectable after the first immunization. Maximum titers of antibody (8.86+/-0.19 ng/ml of serum) were attained after the second immunization. In conclusion, the recombinant S protein expressed in mammary cells was able to elicit substantial immunological response against TGEV. This lays the basis for using mammary gland bioreactor generating edible vaccine.     

Immune interferon production by lymphoid cells: role in the inhibition of herpesviruses.

Bovine peripheral blood lymphocytes (PBL) obtained from infectious bovine rhinotracheitis (IBR) virus- and tuberculin-immunized animals produced large quantities of interferon within 24 h of in vitro stimulation by IBR and purified protein derivative antigens. Separation of PBL into populations enriched in T lymphocytes or B lymphocyte provided the antigen-specific step for immune interferon production. A 2- to 10-fold increase in interferon occurred when lymphocytes were combined with autologous macrophages. Although macrophages, even if treated with antilymphocyte serum to remove any contaminating lymphocytes, could produce some interferon, the augmented interferon produced by macrophage-lypmhocyte cultures was not dmpocytes. Direct physical contact between macrophages and lymphocytes was required for the production of enhanced levels of interferon. Antigen-antibody complexes of irradiated virus-infected cells in the presence of antibody were as efficient or better at stimulating interferon than was free antigen. Because IBR virus was inhibited by interferon levels stimulated in cultures by IBR antigen, it was suggested that the local production of interferon by immune cells might play a similar role in curtailing virus dissemination in vivo, thus leading to recovery from disease.     

Interferon production in cocultures between mouse spleen cells and tumor cells: possible role of mycoplasmas in interferon induction

Interferon production was measured in the murine mixed lymphocyte tumor cell interaction (MLTI) using spleen cells of three inbred mouse strains and a number of in vitro grown lymphoma cells. No difference in interferon production was observed when syngeneic or allogeneic combinations were compared. Interferon was not detectable during the first 6 h of culture and reached its maximum after 12--24 h. In contrast, natural killer cell activity of spleen cells against one of these lymphoma lines (YAC-1) was already high after 4h. A cell line that was not susceptible to natural killing (MDAY-D2) induced the same amount of interferon as YAC-1. These findings suggest that natural killer cell activity and interferon induction in the MLTI are not correlated. Several lymphoma cell lines induced interferon in the MLTI whereas some others did not. All lines that were inductive were found to contain mycoplasmas. Furthermore, even the cell-free supernatants of inducing lines contained mycoplasmas and induced interferon. Purified mycoplasmas and membranes thereof were able to induce interferon production in mouse spleen cells. Our data strongly suggest that interferon production in the MLTI is caused by mycoplasmas. This artifact is the relevance for three reasons. First, cocultures between lymphoma cells and lymphocytes are widely studied in immunology. Secondly, contamination with mycoplasmas is extremely common and often goes unnoticed in immunological laboratories. Thirdly, interferon is known to affect profoundly a number of in vitro functions of immunocompetent cells.     

重组EB病毒BHRF1蛋白对裸鼠脾细胞程序性死亡的抑制作用

目的：观察适当浓度的ＥＢＶ－ＢＨＲＦ１重组蛋白（ｒＢＨＲＦ１）抽提液有无拮抗裸鼠脾细胞程序性死亡（ＰＣＤ）作用。方法：用１×１０６ｍｏｌ／Ｌ地塞米松作用１８ｈ、４３℃加热１０ｍｉｎ及无血清培养４８ｈ等方法诱导脾细胞发生ＰＣＤ,预先加入终浓度为２ｍｇ／Ｌ的ｒＢＨＲＦ１蛋白起拮抗作用。结果：未加ｒＢＨＲＦ１的对照组细胞发生典型的ＰＣＤ,即细胞ＤＮＡ的电泳出现ＰＣＤ特征性的梯形（ｌａｄｅｒ）ＤＮＡ降解片段;电镜观察显示核凝缩,但细胞膜及细胞器结构基本保持正常。若与适当浓度的ｒＢＨＲＦ１预培养８ｈ,再用上述方法处理,则因有重组蛋白的保护而未出现ＤＮＡ降解片段及ＰＣＤ的形态学变化。结论：初步显示ｒＢＨＲＦ１具有抑制脾细胞发生ＰＣＤ的功能。