Activity Of Mouse Spleen Cells Research Articles

Immunostimulatory effects of plasmid DNA and synthetic oligodeoxynucleotides.

Nucleic acid immunization is a new vaccination technology. DNA vaccines do not only carry the genetic information for the antigen of interest but also deliver an adjuvant effect due to the presence of immunostimulatory sequences within the plasmid backbone. It is generally assumed that the adjuvant properties of plasmid DNA are equal to those described for oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs. To challenge this hypothesis we have carried out a series of experiments comparing the ability of single- and double-stranded ODN containing CpG motifs to induce the activation of mouse spleen cells. Moreover, we compared the immunostimulatory properties of plasmids that were modified by the addition of two to four CpG motifs. Our results establish that plasmid DNA express their adjuvanticity as either double or single strands, and no differences were observed between modified and unmodified plasmids. On the other hand, the strongest stimulatory ODN sequences lost their adjuvant properties when administered as double-strand DNA. Furthermore, the profile of cytokines induced on spleen cells by plasmid DNA and ODN is different. Strikingly, plasmid DNA induces a moderate synthesis of IL-6 and a strong synthesis of IFN-gamma, whereas stimulation with ODN showed an inverse profile with a higher increase in the synthesis of IL-6 but a moderate increase in IFN-gamma. Finally, in vivo studies were consistent with the results obtained in vitro. Mice immunized with modified or unmodified plasmids encoding the glycoprotein D of HSV showed similar levels of cellular and humoral immune responses.

Biological activities of lipopolysaccharides extracted from porcine vaccine strains.

Lipopolysaccharides (LPSs) were purified from Actinobacillus pleuropneumoniae serotype 2, Bordetella bronchiseptica and Haemophilus parasuis serotype 5, which were used for vaccine production in Japan, by the phenol-water procedure. In SDS-PAGE analysis, A. pleuropneumoniae LPS, as well as Escherichia coli LPS, demonstrated a typical ladder profile of a smooth-type LPS. On the other hand, B. bronchiseptica and H. parasuis LPSs lacked the ladder profiles. It was found that the biological activity of these LPSs was comparable to those of E. coli LPS in terms of activation of the clotting enzyme of Limulus amoebocyte lysate, mitogenic activity of mouse spleen cells, stimulation of TNF-alpha and nitric oxide production, but IL-6 production could hardly be observed in any LPS.

In vitro activation of mouse spleen cells by a lysate of Theileria sergenti-infected bovine red blood cells

A lysate of Theileria sergenti-infected bovine red blood cells induced in vitro proliferation of mouse spleen cells in a 2–3-day incubation period. Experiments using B or T cell-enriched preparations showed that a B cell (B220 + CD3 − population) was responsible for this non-specific proliferation. In the culture supernatant of spleen cells stimulated with the lysate, considerable amounts of interleukin 2 (IL-2) and gamma-interferon (IFN-γ) were revealed after 2 days, followed by production of immunoglobulin with a peak at 4–6 days. Thus, it was suggested that a lysate antigen(s) of T. sergenti-infected red blood cells was able to stimulate mouse spleen cells to produce IL-2 and IFN-γ, which in turn induced the proliferation of B cells and production of immunoglobulin.

Unique palindromic sequences in synthetic oligonucleotides are required to induce IFN [correction of INF] and augment IFN-mediated [correction of INF] natural killer activity.

Thirty-mer single-stranded oligonucleotides, with a sequence chosen from the known cDNA encoding the 64-kDa protein named Ag A or the MPB-70 protein of Mycobacterium bovis BCG and the human cellular proteins such as complement component 1 inhibitor and Ig rearranged lambda-chain, were used to dissect the capability to induce IFN and to augment NK cell activity of mouse spleen cells by coincubation in vitro. Three with the hexamer palindromic sequence as GACGTC were active, whereas two kinds of oligonucleotides with no palindrome were inactive. The oligonucleotides containing at least one of the different palindromic sequences showed no activity. When a portion of the sequence of the inactive oligonucleotides was substituted with either palindromic sequence of GACGTC, AGCGCT, or AACGTT, the oligonucleotide acquired the ability to augment NK activity. In contrast, the oligonucleotides substituted with another palindromic sequence such as ACCGGT was without effect. Furthermore, exchange of two neighboring mononucleotides within, but not outside, the active palindromic sequence destroyed the ability of the oligonucleotides to augment NK cell activity. Stimulation of spleen cells with the substituted oligonucleotide, A4a-AAC, induced production of significant amounts of IFN-alpha/beta and small amounts of IFN-gamma. Augmentation of NK activity of the cells by the oligonucleotide was ascribed to IFN-alpha/beta production. These results strongly suggest that the presence of the unique palindromic sequences, such as GACGTC, AGCGCT, and AACGTT, but not ACCGGT, is essential for the immunostimulatory activity of oligonucleotides.

Synthetic Oligonucleotides with Particular Base Sequences from the cDNA Encoding Proteins of Mycobacterium bovis BCG Induce Interferons and Activate Natural Killer Cells

Thirteen kinds of 45-mer single-stranded oligonucleotide, having sequence randomly selected from the known cDNA encoding BCG proteins, were tested for their capability to augment natural killer (NK) cell activity of mouse spleen cells in vitro. Six out of the 13 oligonucleotides showed the activity, while the others did not. In order to know the minimal and essential sequence(s) responsible for the biological activity, 2 kinds of 30-mer and 5 kinds of 15-mer oligonucleotide fragments of an active 45-mer nucleotide were tested for their activity. One of the 30-mer oligonucleotides, designated BCG-A4a, was active, but the other 30-mer was inactive. All of the 15-mer oligonucleotide fragments were inactive. The BCG-A4a also stimulated the spleen cells to produce interferon (IFN)-alpha and -gamma. An experiment using anti-IFN antisera showed that the NK cell activation by the oligonucleotide was ascribed to the IFN-alpha produced. It was noticed that all of the biologically active oligonucleotides possessed one or more palindrome sequence(s), and the inactive ones did not, with an exception of a 45-mer inactive oligonucleotide containing overlapping palindrome sequences (GGGCCCGGG). These findings strongly suggest that certain palindrome sequences, like GACGTC, GGCGCC and TGCGCA, are essential for 30-mer oligonucleotides, like BCG-A4a, to induce IFNs.

The Effects of 16,16-Dimethyl PGE2 and Phosphodiesterase Inhibitors on Con A Blastogenic Responses and NK Cytotoxic Activity of Mouse Spleen Cells

Prostaglandins function as down regulators of immune responses probably by increasing the concentration of intracellular cAMP. Phosphodiesterase inhibitors, which prevent the breakdown of cAMP, also increase the intracellular levels of cAMP. Prostaglandins and phosphodiesterase inhibitors have both been shown to suppress immune responses in vitro. In this study 16,16-dimethyl PGE2 (dm-PGE2), added in vitro, suppressed the mouse spleen cell concanavalin A (Con A) response by 38% and natural killer (NK) activity by 53%. Addition of the phosphodiesterase inhibitors, theophylline, RO20-1724, or dipyridamole, decreased both the Con A response and NK activity by at least an additional 30%. We also demonstrate that treatment with dm-PGE2 and theophylline in vivo is more effective than either compound alone in inhibiting NK activity of both untreated mice and mice treated with polyinosinic-polycytidylic acid. These studies support the hypothesis that the immunosuppressive effect of dm-PGE2 is mediated by cAMP and suggest that treatment with a combination of dm-PGE2 and phosphodiesterase inhibitors can augment this immunosuppressive effect.

Effect of benzene and its metabolites on natural killer activity of mouse spleen cells in vitro.

The effects of benzene and its metabolites, phenol and hydroquinone, on natural killer (NK) activity in mouse spleen cells in vitro were studied. NK activity was evaluated by the specific release percentage of 51Cr from labeled YAC-1 cells after YAC-1 cells (target cells) were incubated with spleen cells (effector cells) of mice at ratios of effector cells to target cells (E/T) of 100/1, 50/1, and 25/1. Benzene was shown to inhibit NK activity at concentrations of 1 x 10(-5) M-5 x 10(-5) M, phenol at concentrations of 1 x 10(-7)-5 x 10(-5) M and hydroquinone at concentrations of 1 x 10(-6) M-1 x 10(5) M. Phenol and hydroquinone depressed NK activity in a dose-dependent manner. This study demonstrated that NK cell function of mouse spleen was depressed by exposure to benzene and its metabolites in vitro. Hydroquinone and phenol have very potent toxicity for suppression of immunosurveillance.

In vitro Augmentation of Natural Killer Cell Activity and Production of Interferon‐α/β and ‐γ with Deoxyribonucleic Acid Fraction from Mycobacterium bovis BCG

A nucleic acid‐rich fraction extracted and purified from BCG (MY‐1) augmented natural killer (NK) cell activity of mouse spleen cells in vitro, and produced factor(s) which showed anti‐viral activity and rendered normal macrophages cytotoxic towards tumor cells. These cellular responses were induced by the MY‐1 digested preliminarily with RNase, but not by the MY‐1 digested with DNase, indicating that DNA contained in MY‐1 was essential for the responses. The function of the factor to activate macrophages was destroyed by treatment with a small amount of anti‐interferon (IFN)‐γ antiserum or under acidic conditions (pH 2), but not by treatment with anti‐IFN‐α/β antiserum, while the anti‐viral activity was destroyed almost completely by treatment with anti‐IFN‐α/β antiserum. It appears that DNA from BCG stimulated mouse spleen cells in vitro, resulting in augmentation of NK activity and production of IFN‐α/β and ‐γ.

Increase of natural killer activity of mouse lymphocytes following in vitro and in vivo treatment with lithium.

The in vivo and in vitro influence of lithium lactate on mouse natural killer activity was investigated. In vitro exposure of effector-target mixture to graded concentrations of lithium did not substantially modify the natural killer activity of mouse splenocytes, untreated or pretreated with cyclophosphamide. However in vitro treatment of effector splenocytes increased the frequency of NK-percursor cells. The in vivo treatment with lithium lactate greatly increased the natural killer activity in intact mice, whereas it did not improve this cytotoxic function in host immunodepressed by cyclophosphamide. These data suggest that lithium salts produce a modulation of natural killer activity of mouse spleen cells, probably through a mechanism involving the increase of the number of NK-precursors in hosts not subjected to cytotoxic chemotherapy.

Comparative inhalation toxicity of nickel subsulfide to [formula omitted] rats and B6C3F 1 mice exposed for 12 days

Groups of F344 N rats and B6C3F 1 mice were exposed to aerosols of nickel subsulfide (Ni 3S 2) 6 hr/day for 12 days not including weekends. Actual exposure concentrations were within 3% of target (target = 10.0, 5.0, 2.5, 1.2, 0.6, and 0.0 mg Ni 3S 2/m 3). Nickel lung burdens of exposed rats and mice increased linearly with exposure concentration. Two male rats and all mice exposed to 10.0 mg Ni 3S 2/m 3 died before the end of the exposures. Exposure to Ni 3S 2 had no effect on the natural killer cell activity of mouse spleen cells. Lesions in rats and mice related to inhalation of Ni 3S 2 were found in the nasal epithelium, lung, and bronchial lymph nodes. The most extensive lesions were found in the lung and included necrotizing pneumonia. Emphysema developed in rats exposed to 5.0 or 10.0 mg Ni 3S 2/m 3, while fibrosis developed in mice exposed to 5.0 mg Ni 3S 2/m 3. Degeneration of the respiratory epithelium and atrophy of the olfactory epithelium of the nose occurred in rats exposed to as low as 0.6 mg Ni 3S 2/m 3 and mice exposed to 1.2 mg/m 3. Results indicate that inhalation exposure of rats and mice to Ni 3S 2 aerosol concentrations near the current threshold limit value (TLV) for nickel compounds (1 mg/m 3 for Ni metal and roasting fume and dust and 0.1 mg/m 3 as Ni for soluble compounds) can produce lesions in the respiratory tract. Atrophy of lymphoid tissues (spleen, thymus, and bronchial lymph nodes) was found in animals of the highest exposure concentration. Degeneration of the testicular germinal epithelium was also observed in mice and rats that survived 5.0 or 10.0 mg/m 3 exposure concentrations.

Analysis of changes in natural cytostatic and cytotoxic activity of mouse spleen cells after administration of cyclophosphamide and ?-interferon

The authors studied the effects of cyclophosphamide and alpha-interferon on the natural cytostatic and cytotoxic activity of mouse spleen cells on lymphoma cells. The cells were incubated in medium RPMI-1640 with 10% fetal serum and 1% glutamine in the presence of tritiated thymidine. Incorporated radioactivity was estimated by scintillation counting. The natural cytotoxic activity of the cells was determined by the release of chromium 51 from labelled lymphoma cells transplanted in vitro. Pretreatment of spleen cells with leukocytic interferon was found to cause an increase in both cytotoxic and cytostatic activity of these cells. An investigation of the time course of the antimitotic activity of the cells after pretreatment with cyclophosphamide, causing the death of proliferating cells, showed that effectors of natural cytotoxic activity constitute a homogeneous group as regards sensitivity to cyclophosphamide. Cells exerting cytostatic activity in vitro are evidently heterogeneous for sensitivity to cyclophosphamide. Thymus and bone marrow cells were used for comparative purposes.

Polyclonal antibody secretion in mouse spleen cells induced by staphylococcal phage lysate (SPL)

The effects of staphylococcal phage lysate (SPL) on polyclonal antibody secretion, and mitogenic activity of mice spleen cells were studied. 1) SPL, at a concentration of 30 μl/ml augmented mitogenic activity 1.9 times in mouse spleen cell cultures, however no enhancement of mitogenic activity was observed in B cell depleted, T cell cultures and in athymic nude mouse spleen cell cultures. 2) Augmentation of polyclonal antibody secretion by SPL was observed in athymic nude mouse spleen cell cultures, and did not enhance depleted of macrophages, by passage through Sephadex G-10. 3) By injection of 100 μl SPL, in vivo adjuvant activity against sheep red blood cells were augmented 2.4 times. As revealed above, SPL enhance both mitogenic activity and polyclonal antibody secretion of mouse spleen cells, but augmentation of mitogenic activity is not observed by SPL in T cell, or B cell cultures alone, existence of both T cell and B cell is required to mitogenic activity by SPL. Augmentation of polyclonal antibody secretion by SPL needs B cell, T cell and macrophages.

Effect of anthracycline anticancer agents on natural killer (NK) activity of mouse spleen cells

Effect of anthracycline anticancer agents on natural killer (NK) activity of mouse spleen cells

Cyclosporin A inhibits T cell-mediated augmentation of mouse natural killer activity

Cyclosporin A (CSA) in vitro inhibited the spontaneous cytotoxic activity of mouse spleen cells against YAC target cells in a 4 hr 51Cr release assay. While natural killer (NK) cells were inhibited directly by CSA, these suppressive effects were largely reversible by coculture of effector cells for an optimal period with polyinosinic-polycytidylic acid (Poly I:C) or lipopolysaccharide (LPS). In contrast concanavalin A (Con A), in the presence of CSA, was unable to augment NK activity. The supermatant, however, of mouse spleen cells cultured with Con A was fully able to augment the NK activity of freshly cultured spleen cells in the presence of CSA. The results indicate that CSA inhibits NK activity by two distinct mechanisms: a) a direct inactivation of NK cells and b) a suppression of the production or release of an NK-activating factor from T cells, but not B cells or macrophages.

Exogenous additions of prostaglandins variably alter the blastogenic response of B and T lymphocytes from different mice lymphoid organs

Prostaglandins A1 and E2 enhance the 3H-thymidine (TdR) uptake in spleen cell cultures stimulated by two B-lymphocyte mitogens (E. coli lipopolysaccharide (LPS) and dextran sulfate (DXS) at concentrations ranging between 10−8 and 10−6 M. The same prostaglandin (PG) concentrations inhibited concanavalin A (Con A) activation of mouse spleen cells depleted or not from a glass-adherent prostaglandin-producing population of suppressor cells. After fractionation of spleen cells by nylon wool adherence, PGs diminished 3H-TdR uptake by nylon nonadherent (T-enriched) cells cultured in the presence of Con A and enhanced the activation of nylon adherent (B-enriched) cells by LPS. The proliferative response of thymic lymphocytes to Con A was drastically inhibited (70–95% by PGE210−8−10−6M. In cultures of purified bone marrow lymphocytes, PGE2 induced a dose-dependent inhibition of either LPS- or DXS-induced activation reaching a 30–40% inhibition at a PGE2 concentration of 10−6 M. In the spleen, treatment of cells with anti-T sera and complement resulted in abrogation of PG-induced enhancement. Nevertheless, no inhibition of B-cell mitogenesis was observed in the presence of 10−6 PGE2. From these results, it can be concluded that a different sensitivity of the proliferative response of lymphoid cells to exogenous PGs exists, depending on the subset (T or B) affected and/or the organ used as a source of these cells.

Relation of gp70 to spontaneous cytolytic activity of mouse spleen cells

In comparing spleen cells of inbred and congenic mice for spontaneous capacity to lyse cells of the BALB/c leukemia RLmale1 in vitro, we found that the activity of 129 spleen cells was more than double that of 129-Gix- spleen cells. The only known difference between these two strains is that 129-Gix- mice express no known demonstrable gp70 or p30, whereas 129 mice express both these MuLV-related components as mendelian traits not associated with the production of virions. We infer that MuLV-related components at the cell surface are concerned in effector-target interactions leading to cytolysis under the conditions described. Although the congenic strains B6 (Gix-) and B6-Gix+ differ likewise in expression of the type-variant Gix-gp70, both strains express a second type-variant of gp70. The lytic activity of spleen cells of these two strains for RLmale1 cells was equally high, suggesting that involvement in lytic effector-target interactions is common to gp70 molecules in general. When used as targets rather than as effectors 129 spleen cells were more sensitive to lysis than 129-Gix- spleen cells. Pre-exposure to gp70, purified from R-MuLV, rendered splenic effector cells less lytic. Pre-exposure to gp70 also rendered RLmale1 target cells less sensitive to lysis. One explanation of these findings is that both target cells and effector cells express gp70 and also receptors for gp70 and that this is the basis of mutual cellular recognition leading to lysis in the circumstances described.

Modulation of natural cytotoxicity by alloantibodies: V. The mechanism of a selective augmenting effect of anti-H-2 antisera on the natural killer activity of mouse spleen cells

Treatment of mouse spleen cells with specific anti-H-2 antisera augments their natural killer (NK) activity against K562 cells but not against YAC target tumor cells. The same population of natural killer cells was found to lyse K562 as well as YAC target cells, since (a) depletion of YAC reactive NK cells by absorption on YAC monolayers resulted in a concomitant depletion of anti-K562 NK activity of mouse spleen cells, and (b) both K562 and YAC cells could inhibit their own as well as each others lysis in a cross-competition assay. Anti-H-2 antiserum could not induce anti-K562 NK activity in spleen cells previously depleted of NK cells by absorption on YAC monolayers, indicating that alloantiserum does not act by recruiting otherwise nonreactive cells to become cytotoxic toward K562 target cells. In a target-binding assay, K562 binding of NK cells (T-cell-, B-cell-, and macrophage-depleted spleen cells) increased five- to eightfold in the presence of anti-H-2 antiserum whereas YAC cells binding of NK cells was not increased. H-2 antigens per se did not appear to be involved in the alloantisera effect since anti-NK antiserum directed against a non-H-2 antigen selectively expressed on NK cells, showed a similar selective NK enhancing effect. Protein A, a reagent which binds to the Fc region of immunoglobulin molecules, completely blocked the alloantiserum induced augmentation of anti-K562 NK activity, but did not alter basal NK activity. Moreover, the F(ab) 2 fraction of alloantibodies failed to enhance anti-K562 cytotoxic activity of mouse spleen cells, indicating a crucial role for the Fc portion of the alloantibodies attached to the NK cells, in NK augmentation. Utilization of several target cell lines with or without membrane Fc receptors (FcR) revealed that alloantiserum enhanced the lysis of only FcR + target cells. It is proposed that alloantibody-coated NK cells, as a result of a secondary interaction between attached alloantibody and Fc receptors on target cells, interact more readily with the target cells and thereby cause a higher level of lytic activity.

Modulation of natural cytotoxicity by alloantibodies: IV. A comparative study of the activation of mouse spleen cell cytotoxicity by anti H-2 antisera, interferon, and mitogens

Comparisons were made between the characteristics of cytotoxicity activation in mouse spleen cells, induced by specific anti-H-2 antiserum, interferon (IF), interferon inducer (poly (I:C)), and mitogens (concanavalin A (Con A), pokeweed mitogen (PWM)). Important differences were found in the cytotoxicity activation associated with these agents: (a) The cytotoxic enhancing effect of anti-H-2 antiserum was comparatively rapid and was more pronounced in the first 4 hr of the assay, whereas, IF, poly (I:C), and the mitogens were most effective at 20 hr. (b) Anti-IF antiserum could neutrilize the stimulatory effects of IF and poly (I:C) on the cytotoxic activity of mouse spleen cells but had no influence on the stimulatory effects of anti-H-2 antiserum, PWM, and succinyl Con A. (c) The stimulatory effect of anti-H-2 antiserum was target specific and was observed when the K562 cell line was used as the target but not when YAC, P815, or WFu/G1 target cell lines were used. Stimulatory effects of all the other agents were nonspecific and did not depend on the target cells employed. (c) Cellular fractionation studies suggested that the alloantisera and interferon directly activated the natural killer (NK) cells without any participation of T-cells, B-cells, or macrophages. Both T-cells and NK cells, however, contributed to the mitogen-enhanced cytotoxicity of mouse spleen cells. These results indicated that the mechanism by which anti-H-2 antisera induce NK augmentation in mouse spleen cells is distinct from that of interferon and mitogens.

Modulation of natural cytotoxicity by alloantibodies: III. Augmentation of natural killer activity by alloantisera directed against K, D, and I regions of the murine H-2 complex

Natural killer activity of mouse spleen cells toward a human myeloid leukemia cell line, K562, can be enhanced by alloantisera directed against individual antigens in the H-2 region. By using a panel of 13 antisera (8 directed against antigens in the K and D regions and 5 directed against antigens in the I region) and four strains of mice (C57BL/6J, CBA, DBA/2, and A/J) it was found that certain antisera would stimulate target cell lysis by spleen cells only if the antisera had specificity for antigens which were a part of the haplotype represented on the spleen NK effector cells. Anti Ia antisera could stimulate the anti K562 NK activity of nude mouse spleen cells which lack mature T cells. Depletion of B cells and macrophages from nude spleen cells, by passing through a nylon-wool column also did not abolish the effect of anti-Ia antiserum. It appears likely therefore that the anti-Ia antibodies exert this effect directly on NK cells and that Ia antigens may be expressed on NK cells. Since the antisera directed against different antigens in H-2 complex irrespective of subregion specificity (K, D, or I) stimulated the NK activity of mouse spleen cells, the phenomenon offered an interesting method for testing the presence of a given alloantigen on mouse spleen cells. Log-dose response curves for the augmentation of lysis induced by appropriate alloantisera were linear over a dilution range of 1:320 to 1:5120. By using the dose-response curves, potency ratios of two preparations of antisera (directed against antigen 33 of the K region) could be successfully determined. Besides the K562 cell line, many human lymphoblastoid cell lines could also be used as target cells in this assay system.

Brucella Abortus Lipopolysaccharide Is Mitogenic for Spleen Cells of Endotoxin-Resistant C3H/HeJ Mice

Abstract Preparations of lipopolysaccharide (LPS) from rough and smooth strains of Brucella abortus were mitogenic for spleen cells of athymic nude mice, C3H/HeAU mice, and the endotoxin-resistant C3H/HeJ mice. The mitogenic response induced by crude smooth-LPS (f5) was greater than that produced by purified smooth-LPS (f5p); however, the dose-response curves were similar for both preparations. The mitogenic activity of mouse spleen cells to both f5 and f5p was higher than that produced by stimulation with purified rough-LPS. The dose-response curves with rough-LPS were also qualitatively different from those produced with the preparations of smooth-LPS.