Mouse Spleen Cell Suspensions Research Articles

Tumor growth suppression by implantation of an anti-CD25 antibody-immobilized material near the tumor via regulatory T cell capture

ABSTRACT In this study, we designed and synthesized an implantable anti-CD25 antibody-immobilized polyethylene (CD25-PE) mesh to suppress tumor growth by removing regulatory T cells (Tregs). The PE mesh was graft-polymerized with poly(acrylic acid), and the anti-mouse CD25 antibody was then immobilized using the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide reaction. Immobilization of the antibody on the PE mesh was confirmed by immunostaining. The CD25-PE mesh could effectively and selectively capture CD25-positive cells through antigen-antibody interactions when the CD25-PE mesh was incubated with a suspension of mouse spleen cells, including CD25-positive cells. In addition, implantation of the CD25-PE mesh into mice subcutaneously demonstrated the Treg-capturing ability of the CD25-PE mesh with only a weak inflammatory reaction. In tumor-bearing mice, tumor growth was suppressed by subcutaneous implantation of the CD25-PE mesh near the tumor for 1 week. These results suggested that the anti-CD25 antibody-immobilized material could capture Tregs in vivo and inhibit tumor proliferation in a limited tumor-bearing mouse model. Further research is needed to facilitate cancer immunotherapy using implantable anti-CD25 antibody-immobilized material as a Treg-capturing device.

Macromolecular oxidation in anisotonic suspensions of mouse spleen cells

Macromolecular oxidative alterations have been analysed in vitro in anisotonic suspensions of mouse splenocytes. Both hypertonicity and hypotonicity induced the generation of thiobarbituric acid reactive species (TBARS) and carbonylation of the proteins, which took place along with cell death. Addition of antioxidants partially inhibited oxidative changes in isotonic and hypotonic suspensions. Anisotonic shock of mouse splenocytes proved to be an inducer of oxidative stress. The oxidative macromolecular alterations might contribute to pathogenesis of cell death caused by osmotic stress.

Spermine differentially regulates the production of interleukin-12 p40 and interleukin-10 and suppresses the release of the T helper 1 cytokine interferon-gamma.

Polyamines are endogenous immunomodulatory molecules. Recent studies revealed that polyamines suppress the production of proinflammatory cytokines and nitric oxide. In the present study, we investigated the effect of the polyamines spermine, spermidine, and putrescine on the production of interleukin (IL)-12 p40, IL-10, and interferon (IFN-gamma) in mouse peritoneal macrophages and spleen cell suspensions. Spermine, but not spermidine or putrescine, suppressed, in a concentration-dependent manner, the production of IL-12 p40 by lipopolysaccharide (LPS)-stimulated macrophages. The effect of spermine was post-transcriptional, because steady-state levels of messenger ribonucleic acid (mRNAs) for IL-12 (p35 and p40) were not affected. In contrast to its inhibitory effect on IL-12 p40, spermine (0.3-3 microM) augmented IL-10 production. The down-regulation of IL-12 p40 by spermine was independent of enhancement of IL-10 by this agent, for spermine retained its ability to suppress IL-12 production in peritoneal macrophages obtained from IL-10-deficient mice. The alterations in cytokine production by spermine did not involve an effect on early intracellular pathways of LPS signal transduction, including the p38 or p42/44 mitogen-activated protein kinases, or the c-jun terminal kinase. In spleen cell suspensions, spermine suppressed the release of IFN-gamma induced either by LPS or anti-CD3 antibody. In summary, spermine exerts anti-inflammatory effects by suppressing IL-12 and IFN-gamma and by augmenting the production of IL-10.

Immunoregulatory properties of hexapeptide isolated from porcine bone marrow cell culture

Myelopeptide 1 (MP-1) is hexapeptide originally isolated from porcine bone marrow cell culture. It was synthesized and its immunoregulatory properties were studied. MP-1 caused a 1.5–2-fold dose-dependent increase of antibody production in the culture of mouse immune lymph node cells. It abolished Con A induction of T suppressors in the suspension of mouse spleen cells and counteracted the inhibitory effect of T suppressors on antibody production. The inoculation of MP-1 (1·10 −9 g/mouse) to mice two weeks after their γ-irradiation (2 Gy) resulted in an increase of antibody production up to 80.2 ± 15.5% as compared to that in the irradiated control 37.6 ± 12.0%. Immunofluorescent analysis revealed the specific binding of MP-1 with receptors on the target cells in the suspension of mouse spleen cells. It is supposed that MP-1 participates in the immunoregulatory processes in the living organism.

The Effect of Virus Particle Size on Chemiluminescence Induction by Influenza and Sendai Viruses in Mouse Spleen Cells

Suspensions of orthomyxo- and paramyxoviruses are composed of pleomorphic particles ranging from large filaments to small spheres. Influenza and Sendai viruses were separated according to size by gel filtration and the induction of luminol-dependent chemiluminescence (CL) by particles of similar size was studied in suspensions of mouse spleen cells known to contain phagocytes. CL reflects the generation by the cells of reactive oxygen species. CL induction decreased with particle size for both viruses. Compared with small spheres, large influenza filaments were approximately 10 times as efficient in activating cellular light emission while the ratio between large and small Sendai viruses was 3:1. Small Sendai virus particles were also less efficient in lysing red cells and had lower neuraminidase activity. By contrast, with influenza virus, only neuraminidase and not the hemolytic activity decreased with the virus size. When influenza virus filaments were broken into smaller particles by sonication, the capacity to induce chemiluminescence dropped markedly while the hemolytic and hemagglutinating activities increased and neuraminidase activity remained unaltered. These results suggest that the presentation of influenza virus hemagglutinin and neuraminidase glycoproteins in a large particle, leading to extensive receptor crosslinking, may be an important factor in the efficient activation of CL by filamentous influenza virus. We suggest that radical generation as reflected in cellular CL may relate to the toxic in vivo effects that contribute to the pathogenesis of influenza and infections with paramyxoviruses.

Effect of hydrocortisone on superoxide radical production by phagocytic spleen cells

Superoxide radical production by mouse phagocytic spleen cells was shown to be essentially increased 2 hours following intraperitoneal injection of hydrocortisone acetate at a dose of 50 mg/kg body weight. The addition of hydrocortisone to the suspension of mouse spleen cells has resulted in linear dependence of hormone concentration in the incubation medium on the maximum rate of superoxide radical production. The mechanism of hydrocortisone stimulating effect on the activity of plasma membrane-located NAD(P)H-oxidase of phagocytic cells is being discussed.

Interleukin induction of non-specific suppressor cells

We analysed the cellular and molecular signals involved in the induction of non-specific T suppressor (Ts) cells generated in mouse spleen cell suspensions after 3-4 days' culture in the absence of conventional antigen. Using an in vitro model of primary antibody response as a suppressor assay, we showed that purified T cells were unable to generate suppressive activity. This suppression was fully restored when T cells were cultured with macrophages. In this case, Ts-cell generation was markedly increased by the addition of indomethacin suggesting a negative effect of prostaglandins in this phenomenon. Macrophages could be replaced by semi-purified IL1 as well as by recombinant IL2. Thus, in conditions of severe macrophage depletion, IL1 and/or IL2 are fully capable of inducing Ts-cell generation.

Specific binding of cholesterol to chromatin prepared from mouse spleen cells.

Mouse spleen cell suspensions were incubated with tritiated cholesterol for various time intervals. The chromatin of these cells was then isolated, washed with Triton X-100, and fractionated on Sephadex columns. It was found that cholesterol binds specifically to the chromatin. The binding was saturated after 45 min of incubation and also displayed characteristics typical of dose-dependent binding. The number of cholesterol molecules bound per nucleus was estimated to be on the order of 10 000. Oxygenated sterols, such as 25-hydroxycholesterol and 7-ketocholesterol, did not compete for the binding with [3H]cholesterol if added in 20-fold molar excess to the incubation medium of the cells. Chromatographic analyses on Sephadex columns displayed a distinct peak of radioactivity. The protein-sterol complex had an apparent molecular weight of 180 000 +/- 27 000. Using extensive digestion with DNase I (EC 3.1.21.1) it could be concluded that DNA, binding to the complex, did not influence the estimate of the molecular weight, whereas digestion with pronase or treatment with sodium dodecyl sulfate destroyed the complex. Additional experiments using sucrose density gradients (5-20%) showed also, that [3H]cholesterol was bound to chromatin by one or several proteins.

Interferon induced in mouse spleen cells by Staphylococcus aureus

Interferon was produced in suspensions of mouse spleen cells treated with Staphylococcus aureus preparations (killed bacteria, culture supernatants, or purified enterotoxin) under a variety of cell culture conditions. The lysate of S. aureus was found to induce high levels of interferon (10 3.1 to 10 4.3 RU/ml) within 72 hr. The crude interferon was concentrated and partially purified by either ammonium sulfate precipitation or adsorption to silicic acid and elution by ethylene glycol-containing buffer. Sequential precipitation with 50 to 80% saturated ammonium sulfate resulted in a three- to seven-fold purification with 60% recovery of activity. Adsorption to silicic acid resulted in a 25- to 80-fold purification with 77% recovery. This material was further analyzed by gel filtration. The antiviral activity induced by S. aureus-treated spleen cells was characterized as due to interferon. Furthermore, the inhibitor was acidlabile and not neutralizable by antiserum against NDV-induced L-cell interferon, thus exhibiting properties of immune (γ) interferon. The partially purified interferon was used to prepare an antiserum in rabbits. This antiserum was able to neutralize mouse interferon induced by several T-cell mitogens, by antigens, and by mixed lymphocyte cultures, while remaining inactive against interferons induced in vitro by viruses or in vivo by Brucella abortus.

NK cell-target interactions: approaches towards definition of recognition structures.

NK cells lyse an uncommonly wide range of cell types, implying either that they (the NK cells) have clonally distributed receptors each of which is capable of interacting with a very limited number of cell types or, alternatively, that susceptible target cells share a common characteristic. A number of experimental approaches have suggested that the cytotoxic 'specificity' of NK cells is not clonally distributed. Thus, clones of NK cells, established from mouse spleen cell suspensions, showed no greater restriction in the spectrum of target cells which they could lyse, than did the parent spleen cell populations from which they were derived. It seems likely therefore that the wide range of target cell types that can be lysed by NK cells share common cell surface characteristics which render them susceptible. As lysis results from membrane-membrane interactions, it seemed logical that a search for 'hallmarks' of NK susceptibility should begin with a detailed examination of the plasma membrane of susceptible cells. Analysis of one pair of lymphoma cell variants, selected on account of their markedly different susceptibility to NK cells, suggests that cell surface glycoconjugates may be of significance in determining those effector cell-target cell interactions that lead to lysis. This review outlines attempts to characterize such glycoconjugates.

Sendai virus stimulates chemiluminescence in mouse spleen cells

Sendai virus stimulates chemiluminescence within a few seconds after it is added to a suspension of mouse spleen cells. Virus rendered non infectious by irradiation with ultraviolet light induces a similar burst of chemiluminescence. Heating or pronase treatment of the virus abrogate this reaction, as does sonication of the cells before the addition of the virus. The ability of the virus to stimulate chemiluminescence is correlated with its hemagglutination, neuraminidase, cell fusion and hemolytic properties. It is suggested that Sendai virus-induced chemiluminescence is initiated by the interaction of the virus envelope spike glycoproteins with the cell membrane.

In Vitro Antibody Response to Influenza Virus

Abstract A good secondary IgG response to the hemagglutinin (HA) of influenza virus has been obtained in vitro in Marbrook-type cultures of influenza-primed mouse spleen cell suspensions stimulated with inactivated influenza virus. Anti-HA antibody was quantitated by a solid phase radioimmunoassay (RIA) by using purified HA as substrate. The T dependence of this secondary response was shown by depletion of T cells and reconstitution with a source of primed or unprimed T cells. The help given by T cells primed to the homologous virus was many times greater than that given by unprimed T cells. Although the latter was significant. The system described will allow investigation of the specificity requirements of helper T cells engaged in the anti-HA response.

High gradient magnetic separation of erythrocytes

The high gradient magnetic separation technique has been applied to separate paramagnetic erythrocytes from a cell suspension that also contained diamagnetic cells. Paramagnetism was induced in the red blood cells by oxidizing the iron atoms in the cell hemoglobin to the ferric state (methemoglobin). Diamagnetic cells were either untreated erythrocytes, containing oxyferrohemoglobin, or leukocytes in a suspension of mouse spleen cells. Cell suspensions were passed through a column containing 40 micron diameter stainless steel wire in a high magnetic field (33 kG). The paramagnetic cells were retained on the surface of the wire while the diamagnetic cells passed through. Elution of the paramagnetic cells was accomplished by removing the column from the magnet, in effect turning off the field.

Evidence for control of complement receptor rosette-forming cells by alpha and beta-adrenergic agents.

PHARMACOLOGICAL agents such as dibutyryl cyclic AMP1,2, cholera toxin3,4 and β-adrenergic stimulants5 have an effect on antibody production which can be inhibitory or enhancing, depending on the dose used and the time of addition of the agent. Such stimulants are known to inhibit the human T-lymphocyte receptor for sheep erythrocytes6,7 and the murine lymphoma Fc receptor8, and it has been shown that the complement receptor on B lymphocytes has an important role in the cellular events leading to antibody production9,10, thus emphasising the need to clarify its nature. We report here that the number of complement receptor rosette-forming cells (EAC–RFC) in suspensions of mouse spleen cells can be decreased by α-adrenergic stimulants and increased by β-adrenergic stimulants, respectively.

Role of T and Adherent Cells in <i>in vitro</i> 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.

The rapid onset of inhibition of antibody‐forming cell proliferation upon an increase in density of cellular suspensions cultured in vitro

It was shown previously that the development of primary immune response in vitro in the Mishell-Dutton system was strongly inhibited by increased cell densities (Gurvich et al., Immunology 1975. 28: 271). In the present work, the rate of onset of this inhibition was studied in C57BL/6 mouse spleen cell suspensions during the phase of exponential increase of antibody-forming cells (AFC) after 3 days' cultivation in vitro with the antigen. It has been shown that the increase in AFC number is very soon inhibited following an increase in the cellular density, the inhibition already being evident within 2 or 4 h. Parallel reduction in incorporation of radioactive precursors in DNA and especially RNA is observed, whereas inhibition of protein synthesis was delayed.

Induction of megakaryocyte colonies with platelet formation in vitro.

COLONY culture methods utilising semi-solid media which facilitate extensive cellular differentiation in vitro have proved invaluable for the quantitative investigation of granulocytic and erythrocytic progenitors and the effector substances to which they respond1,2. Colonies of megakaryocytes have been produced from suspensions of mouse bone marrow or spleen cells in agar containing lymphocyte-conditioned medium3. But these megakaryocytes did not reach the stage of maturation at which significant platelet production could be demonstrated. We now report the development of megakaryocyte colonies with extensive platelet production in plasma cultures seeded with mouse bone marrow cells and containing either sheep or human erythropoietin in the medium. The proportional relation observed between the number of colonies produced and the number of bone marrow cells plated also provides the basis of a quantitative assay method for megakaryocyte progenitors in murine haemopoietic tissues.

Enrichment in B cells after separation of lymphocytes from peripheral blood

Mouse spleen and blood cell suspensions were subjected to three of the techniques routinely used for separating lymphocytes from peripheral blood: gelatin sedimentation, dextran sedimentation and the Ficoll-Triosil technique. B cells were distinguished by surface labelling with fluorescein-conjugated anti-mouse immunoglobulin. Studies were made on the recovery of different classes of mononuclear cell in the fractions from separation. The results suggest that: (a) the fraction which would normally be collected if the techniques were being used to prepare blood lymphocytes was considerably enriched in B cells, (b) the more rapidly sedimenting fractions were relatively enriched in cells bearing neither immunoglobulin nor the theta antigen, (c) there was considerable destruction of T cells.

Mode of action of a supernatant activity from T-cell cultures that nonspecifically stimulates the humoral immune response.

The mode of action of "allogeneic supernatant" (the culture supernatant of a 24-hr mixedlymphocyte reaction), has been studied. This factor stimulates the response of spleen cell cultures depleted in thymus-derived lymphoid cells (T-cells) to antigens that elicit a thymus-dependent response. We used a limiting dilution analysis, in which the frequency and size of response of individual bone-marrow-derived lymphoid cells (B-cells) could be measured. In confirmation of other reports, the occurrence of B-cells responding to antigen under different conditions was shown to follow a Poisson distribution in mouse spleen cell suspensions. Allogeneic supernatant increased responses to thymus-dependent antigens, both by increasing the frequency of B-cells whose response is initiated and by increasing the numbers of antibody-forming cells obtained from each responding B-cell. Two fractions were obtained by dialysis of the supernatant. The nondialyzable fraction contained factors able to increase both the frequency of B-cells responding to sheep erythrocytes, and the size of the responding unit. The dialysate contained factors that were only able to increase the numbers of antibody-forming cells obtained per responding B-cell from B-cells whose response had already been initiated by antigen-specific T-cells. Since the nondialyzable factors were active in the absence of detectable functional T-cells, it was concluded that these factors, produced by T-cells, might represent one mechanism whereby T-cells cooperate with B-cells in the initiation or development of a humoral immune response.

Inhibitory and stimulatory effects of concanavalin A on the response of mouse spleen cell suspensions to antigen. II. Evidence for separate stimulatory and inhibitory cells.

The addition of concanavalin A to mouse spleen cell suspensions can either inhibit or stimulate the immune response to heterologous erythrocytes according to the experimental conditions. Evidence is presented which is incompatible with the hypothesis that these two effects are mediated by a single cell and which favors the hypothesis of separate inhibitor and stimulatory cells.