Stimulation Of Mouse Spleen Cells Research Articles

Studies of macrophage function in murine systemic lupus erythematosus. 4. failure to reverse the defect in fc-mediated phagocytosis and binding by in vitro stimulants or prostaglandins

Previous studies have shown that resident peritoneal macrophages from mice with systemic lupus erythematosus (SLE) show defective Fc-mediated phagocytosis and binding of opsonized sheep erythrocytes (EA) in vitro. Possible causes of this defect in (NZB X NZW)F1 (B/W) mice were investigated. These included a maturational block in peritoneal macrophage differentiation, the production by peritoneal cells of a factor which inhibits Fc receptor expression and phagocytosis, and an abnormal response by macrophages of autoimmune mice to prostaglandins. Resident peritoneal macrophages of B/W mice did not show a maturational block since incubation with either (a) differentiating agents such as 4 beta-phorbol 12 beta-myristate 13 alpha acetate or retinoic acid, or (b) lymphokine (LK), prepared by Con A stimulation of mouse spleen cells, failed to enhance Fc-mediated phagocytosis and binding by B/W cells relative to controls. However, LK from B/W and B6AF1 cells stimulated Fc-mediated phagocytosis and binding by bone-marrow (BM)-derived macrophages of CBA/H mice; B/W LK also stimulated BM cells from B/W mice. Peritoneal cell supernatants did not inhibit phagocytosis of Fc receptor expression by BM-derived macrophages in vitro. Prostaglandin E treatment of peritoneal or BM-derived macrophages in vitro failed to restore decreased phagocytosis and binding of EA induced by culture in indomethacin and failed to stimulate phagocytosis by untreated cultures. The reason for the observed defect remains obscure.

An inhibitor of interferon action: II. Biological properties of the IFN-gamma-associated inhibitor of interferon action.

Previously an inhibitor of interferon action had been isolated from mitogen stimulated mouse spleen cells. This inhibitor was associated with the IFN-gamma molecule and might possibly represent an altered IFN-gamma molecule which can no longer induce an effective antiviral state. This report further investigates the biological properties of this inhibitor. Inhibitor activity is independent of the virus used in the interferon assay. Inhibitor activity has also been found to be species specific. Mouse inhibitor does not inhibit the antiviral activity of either human IFN-gamma or human IFN-beta. However, inhibitor production is not limited to the mouse system. Inhibitor is also present in preparations of human IFN-gamma. The inhibitor does not appear to directly compete with IFN-gamma for a specific cell surface receptor since inhibitor activity is independent of interferon concentration. The presence of inhibitor allows a unique, albeit reduced level of antiviral protection to develop. Increasing concentrations of interferon do not increase the level of antiviral protection allowed by the inhibitor. This inhibitor of interferon action may represent a natural mechanism whereby IFN-gamma-induced effects are regulated in vivo.

LY-2+ effectors cytotoxic for syngeneic tumor cells: generation by allogeneic stimulation and by supernatants from mixed leukocyte cultures.

The present study was aimed at gaining insight into means by which stimulation of mouse spleen cells with allogeneic normal cells in mixed leukocyte cultures (MLC) can result in the generation of effector cells cytotoxic for syngeneic tumor or transformed cells. Stimulation of lymphocytes from BALB/c or C3H mice for 5 days with cells from mice of every allogeneic strain tested, in medium containing mouse serum and lacking xenogeneic serum, resulted in the activation of effectors cytotoxic for syngeneic cells transformed spontaneously or by SV40, polyoma or adenovirus. In each experiment, all of the syngeneic transformed cell lines, as well as clones derived from these lines, were lysed to the highest degree by effectors obtained from the same culture, and therefore stimulated with cells from the same allogeneic strain. Although the particular allogeneic sensitizing strain that induced the highest cytolytic activity varied between experiments, effectors obtained from the culture with the highest cell recovery always exhibited the greatest cytotoxicity against all the syngeneic transformed cells and clones. Lysis was mediated predominantly by Ly-2+ effectors; total lytic units of cytotoxicity recovered after treatment with monoclonal anti-Ly-2 antibody and complement (C) were reduced by 85 to 90% compared to cells treated with C alone. Lysis of syngeneic tumor cells by the allosensitized effectors in cytotoxicity assays was not inhibited by the addition of unlabeled "blocking" lymphocytes from the allogeneic strain used for sensitization. In addition, it was found that lymphocytes cultured without stimulating cells for 5 days in medium supplemented with supernatants from secondary MLC that are known to contain high levels of lymphokines, mediated high levels of cytotoxicity on all the transformed cells tested, but lacked detectable cytotoxic activity for syngeneic or allogeneic Con A blasts. The MLC supernatant-activated effectors that lyse the transformed cells are phenotypically CTL, because treatment with anti-Ly-2 and C reduced lytic activity by approximately 75%. Taken together, these findings suggest that the generation in MLC of Ly-2+ effector cells cytotoxic for syngeneic transformed cell lines might not be due, in some cases, to lymphocyte responses to particular alloantigens on the stimulating cells that are cross-reactive with "alien" histocompatibility antigens on transformed cells, but rather is due to effector cell activation by lymphokines produced during allogeneic stimulation.

Studies on a new lymphocyte mitogen from pollen aqueous extract: induction of proliferation in human and rodent lymphocytes.

Fraction III (Fr III) mitogen was isolated from the whole aqueous extract (WAE) of plane-tree pollen by Con A-Sepharose chromatography and gel filtration. It stimulated mouse spleen cells, and peripheral blood lymphocytes from plane-tree non-sensitive patients or from cord blood. A 6-day culture gave the best response. The proliferation was not abolished by washing the cells after a short pulse of Fr III given in the initial step but was inhibited by alpha-D-methylmannoside. These results suggested that Fr III bound to a specific site onto spleen cells. This mitogen was not immunogenic in mice and did not bind to human IgE. It induced the proliferation of T lymphocytes in presence of accessory cells and was a polyclonal activator.

Flow cytometric analysis of the effect of phenytoin and its major metabolite on mitogen stimulated mouse spleen cells

The in vitro immunopharmacological effects of phenytoin (PHT) and its major metabolite 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) were investigated by flow cytometric analysis of the DNA content of mitogen stimulated mouse spleen cells. The qualitative effects of PHT and HPPH were similar in concanavalin A stimulated mouse spleen cells with both compounds causing an increase in the percentage of S phase cells. The data suggests that this effect is due to an augmentation of cell cycling as demonstrated by the significant increase in 4N cells in PHT treated cultures relative to control cultures following colcemid treatment. A PHT time course study revealed an increase in S phase cells and a subsequent increase in 4N cells. PHT had no significant effect on congenitally athymic nude mouse spleen cells stimulated with lipopolysaccharide (LPS) except at the highest concentration tested (80 μg/ml) where a depression of cell cycling was observed. HPPH caused a colcemid-like accumulation of 4N cells in the LPS stimulated nude mouse spleen cell cultures. PHT and HPPH were found to be effective in enhancing cell cycling in cultures containing a significant population of T-cells stimulated with a T-cell mitogen whereas an inhibitory effect was observed in cultures without T-cells stimulated with a B-cell mitogen. The capacity of PHT to enhance the mitogenic action of concanavalin A may relate to its capacity to induce immunologic abnormalities and lymphadenophathy in humans.

Gamma interferon (IFN gamma) and IFN alpha/beta suppress murine myeloid colony formation (CFU-C)N: magnitude of suppression is dependent upon level of colony-stimulating factor (CSF).

The effect of different types of interferon (IFN) on macrophage and granulocyte colony formation (CFU-C) in vitro was studied using bone marrow cells from C57BL/6 mice. CFU-C formation was measured at 7 days using a standard methylcellulose culture system with L-cell conditioned medium (LCCM) as a source of colony-stimulating factor activity (CSF), IFN gamma was obtained from staphylococcal enterotoxin A- (SEA) stimulated mouse spleen cells and was partially purified (50-fold) using affinity chromatography. IFN alpha/beta was obtained from Newcastle disease virus-infected mouse L-cells and was partially purified (286-fold) using an AcA 202 column. IFN gamma inhibited CFU-C formation in a dose-dependent manner and was 100 times more inhibitory than IFN alpha/beta. IFN gamma completely suppressed CFU-C formation with as little as 15 units of antiviral activity. In contrast, 1000 to 2000 units of antiviral activity of IFN alpha/beta was required for complete suppression of CFU-C formation. The ability of IFN gamma to inhibit CFU-C formation was completely abolished after pH 2 treatment (24 hr), heat treatment (60 degrees C, 2 hr), or treatment with a specific antiserum against IFN gamma, which indicated that IFN gamma was the mediator of CFU-C inhibition. When IFN gamma and IFN alpha/beta were mixed together and added to bone marrow cells, they had a synergistic effect on the suppression of CFU-C formation. The level of inhibition mediated by both IFN gamma and IFN alpha/beta was totally dependent upon the amount of CSF present in the culture system; increasing the level of CSF could completely overcome the IFN suppressive effect. These results suggest that IFN gamma and IFN alpha/beta may have regulatory roles in hematopoiesis.

Differential effect of low molecular weight alcohols on the Con A stimulation of mouse spleen cells

Incorporation of [ 3H]thymidine ([ 3H]TdR) into concanavalin A (Con A)-stimulated murine splenocytes was found to be enhanced by addition of certain concentrations of ethanol, 2-propanol and acetone. The alcohol/acetone-induced enhancement of the Con A response was found to be accompanied by an increase of the percentage of living cells as assessed by trypan blue exclusion. Concentrations of ethanol and 2-propanol which caused maximum [ 3H]TdR uptake in Con A cultures were also found to lead to higher percentages of aggregated cells than in comparison to Con A cultures without alcohol. The data suggest that alcohols in certain concentrations are capable to achieve optimum Con A stimulation.

Production of mouse lymphotoxin by phytohemagglutinin-stimulated spleen cells requires two cell fractions.

The appearance of lymphotoxin in the culture fluid of phytohemagglutinin (PHA)-stimulated mouse spleen cells required two cell fractions that were separated by adherence to plastic. Upon stimulation with PHA, neither cell fraction alone produced significant amounts of lymphotoxin; however, when the cell fractions were combined and then stimulated with PHA, full activity was produced. Cytotoxic activity was not fully restored by combining PHA-stimulated cultured fluids from adherent and nonadherent cell fractions. This indicated that the cytotoxic activity was not the result of two factors, one produced by each cell fraction, that acted on the target cells, but rather, two cells interacted to produce lymphotoxin. Treatment of the unfractionated spleen cells with monoclonal anti-Thy1.2 and complement before PHA stimulation greatly reduced the production of lymphotoxin and indicated that at least one of the cells was a T cell. Lymphotoxin production was partially restored by the addition of nonadherent cells to the anti-Thy1.2-treated cells, suggesting that the T cell was nonadherent. Treatment of unfractionated cells with either silica or carrageenan had no effect on the subsequent production of lymphotoxin by PHA, suggesting that the adherent cell was not actively phagocytic.

Soluble cytotoxic factors and the mechanism of NK cell mediated cytotoxicity.

Soluble cytotoxic factors from mouse spleen cells have been shown to selectively lyse NK sensitive target cells. Lysis of target cells is assessed by trypan blue uptake or 51Cr--release assay in a 16-48 hour assay. The possible role of such natural killer cytotoxic factors (NKCF) in the mechanism of natural killer cell-mediated cytotoxicity (NKCMC) has been examined. Several lines of evidence are presented which indicate that there exists a strong correlation between lysis by NKCF and lysis in NKCMC. For instance, (1) NKCF are generated following stimulation of mouse spleen cells with NK sensitive targets; (2) Lysis of NKCF is selective for NK sensitive targets and is species specific; (3) Mice with poor NK activity, such as Bg/Bg mice, produce poor NKCF: (4) There is concomittant inhibition of NKCMC and NKCF activities by blocking RAT serum; and (5) Several known characteristics of the mechanism of NKCMC are shown to be shared in the NKCF system. Based on these findings, we propose a model for NKCMC in which lysis by NK effector cells is the result of multiple steps, namely target binding to an NK effector cell, activation of the lytic mechanism, and involvement of NKCF to mediate lysis. Accordingly, for targets to be NK sensitive, they ought to be able to interact and bind with NK effectors, activate the NK cells, bind NKCF, and be sensitive to the NKCF lytic activity.

Mechanism of T-cell help in the immune response to soluble protein antigens II. Reconstitution of primary and secondary in vitro immune responses to dinitrophenyl-carrier conjugates by T-cell-replacing factor.

Spleen cells of dinitrophenyl keyhole limpet hemocyanin (DNPKLH) primed and boosted mice produced a nonantigen-specific helper factor upon in vitro challenge with DNPKLH. This helper factor displays all of the biological characteristics so far described for TRF produced by allogeneic or Concanavalin A stimulation of mouse spleen cells. It restores the primary anti-SRBC response in nude spleen cultures following the same kinetics of action as T-cell-replacing factor (TRF). Conversely, TRF restores the primary in vitro immune response of nude spleen cultures to DNPKLH. TRF also restores the secondary anti-hapten IgG response of T-cell-deprived spleen cell cultures derived from DNPKLH primed and boosted mice. Here the need for carrier specificity is fully overcome. The data therefore suggest that TRF, as a nonantigen-specific maturation signal, is involved in the primary and secondary immune responses to both particulate and soluble antigens.

Characterization of mouse lymphocyte-derived chemotactic factor

Chemotactic factor was produced in vitro by antigen or mitogen stimulation of mouse spleen cells. Activity was eluted from Sephadex columns in a major peak at 38,000 daltons. Small amounts of activity sometimes occurred in the 13,000 molecular weight region. Total recovery in the eluted fractions ranged from 30 to 60% of the applied material. The molecular weight of mouse lymphocyte-derived chemotactic factor (LDCF) is different from that of human, guinea pig, and chicken, all of which have reported molecular weights of 12,500 to 15,000. Mouse LDCF was eluted from DEAE columns at a specific conductance of about 15 mmho/cm, which indicates an apparent net charge different from chemotactic factor produced by stimulated human peripheral blood leukocytes. Thus, among lymphocyte-derived chemotactic factors hitherto described, the 38,000 MW molecule produced by mouse spleen cells is unique.

Endotoxin-stimulated spleen cells: dissociation between DNA synthesis and IgM production at the cellular level.

LPS stimulation of mouse spleen cells in the presence of Hu resulted in almost total suppression of [3H]thymidine incorporation without affecting the percentage of cells induced to produce IgM. Utilizing a method which permitted the simultaneous measurement of IgM production and [3H]thymidine incorporation in individual cells, it was demonstrated directly that LPS stimulation of IgM production can occur in the absence of DNA synthesis.

Dual pathway of B lymphocyte differentiation in vitro.

Direct visualization of the events resulting from LPS stimulation of mouse spleen cells in vitro was achieved by characterizing the cells during four days of culture for morphology, Ig and theta surface markers and autoradiography after [3H] thymidine uptake. The changes observed were related to biochemical parameters such as incorporation of [3H] thymidine into DNA, Ig biosynthesis and secretion. Two pathways of B lymphocyte differentiation were observed: a) the generation of a large number of small B lymphocytes with high density of surface Ig but no internal pool detectable by immunofluorescence, and b) the maturation of a very small proportion of cells with a large intracellular pool and the ability to secrete Ig. Both cell types arise from dividing blast cells, either physically separated or traced by pulse chase experiments with [3H] thymidine. We discuss whether this duality is caused by the triggering of different B cell subpopulations at different developmental stages, preprogramed to one or the other pathway or whether the final direction of development depends on the microenvironment of individual dividing cells.

Endotoxin-Stimulated Spleen Cells: Mitogenesis, the Occurrence of the C3 Receptor, and the Production of Immunoglobulin

Abstract Endotoxin (LPS)-stimulated mouse spleen cells were investigated in an attempt to correlate three markers for B lymphocytes: 1) LPS-induced blastogenesis, 2) rosette formation via the C3 receptor site, and 3) immunoglobulin production. After 24 hr stimulation 86% of LPS-induced blast cells formed rosettes indicating that these cells were B lymphocytes. Subsequently there was a progressive decrease in the percentage of rosette-forming blast cells to a low of 39% at 72 hr. The percentage of cells producing IgM increased progressively to a maximum of 54% after 72 hr of LPS stimulation These data indicate that at given times correlations between the three markers vary, and that no one of the three alone allows a strict categorization of cell types. The data presented are consistent with the hypothesis that LPS is a mitogen for B lymphocytes, although the possible involvement of other cell types was not ruled out.

Mitogenic properties of lectin and its chemical dervatives.

A mitogenic lectin that has a carbohydrate-binding specificity similar to that of concanavalin A (Con A) can be isolated from pea seeds. Chemical modification (succinylation, acetylation, or treatment with the diazonium salt of sulfanilic acid), changes its biological properties. In mitogenic stimulation of mouse spleen cells and in hemagglutination, the differences between the chemically modified pea lectin and the native molecule are similar to those observed between succinyl-Con A and native Con A. However, whereas chemical modification converts tetrameric Con A to a dimeric molecule, similar treatments of the pea lectin do not change its quaternary structure. The results of binding studies of the pea lectin and its derivatives to mouse spleen cells suggest that the differences in biological activities may be explained by a reduction in binding affinity of the pea lectin for glycoproteins on the spleen cell surface after chemical modification.

DNA synthesis by cultured lymphocytes: A modified method for measuring 3H-thymidine incorporation

A rapid, convenient and inexpensive method for harvesting lymphocyte cultures and measuring the incorporation of 3H-thymidine into trichloroacetic acid precipitable material has been developed. The basic principle is to adsorb the entire contents of a microculture well onto the cotton applicator portion of a Q-tip, precipitate the DNA, wash away unincorporated 3H-thymidine, and count the remaining 3H in a mixture of scintillation fluid plus detergent. Data presented for mixed lymphocyte cultures between allogeneic rat lymph node cells, mixed lymphocyte cultures of human peripheral blood lymphocytes, Con A stimulated mouse spleen cells, and PHA stimulated mouse spleen cells show the method to be highly reproducible with standard deviations of less than 15% of the mean for quadruplicate mixed lymphocyte cultures and in most cases less than 5% of the mean for duplicate mitogen stimulated cultures. This culture system also gives positive values for PHA stimulated DNA synthetic responses of mouse spleen cells cultured in RPMI-1640 plus penicillin and streptomycin but without exogenous serum.

Concanavalin A derivatives with altered biological activities.

Chemical derivatization of tetrameric concanavalin A (Con A) with succinic anhydride or acetic anhydride converts the protein to a dimeric molecule without altering its carbohydrate-binding specificity. At low concentrations, the dose-response curves for the mitogenic stimulation of mouse spleen cells by native Con A and succinyl-Con A are similar. Above lectin concentrations of 10 mug/ml, however, the response to Con A is diminished, while that for succinyl-Con A does not decrease until much higher doses are reached. We have attributed this difference mainly to the higher rate of cell death induced by the native Con A molecule. Con A also shows a greater capacity than succinyl-Con A to agglutinate sheep erythrocytes and to inhibit cap formation by immunoglobulin receptors on spleen cells. Moreover, at low concentrations, Con A induced its glycoprotein receptors to form caps, but succinyl-Con A did not induce cap formation. Addition of antibodies directed against Con A to succinyl-Con A bound on cells restored the properties of agglutination, inhibition of immunoglobulin receptor cap formation, and induction of cap formation by Con A receptors. Similar results have been obtained for acetyl-Con A. These data suggest that the altered biological activities of succinyl-Con A and acetyl-Con A are attributable to their reduced valence.

Mixed leukocyte culture with mouse spleen cells in a serum-free medium.

SummaryFollowing experiments which suggested that apparent stimulation of mouse spleen cells by fetal calf serum (FCS) was masking specific blastogenic effects in the mixed leukocyte culture (MLC), cultures of CBA and BALB/c spleen cells were set up in RPMI-1640 medium, containing FCS at 5, 4, 3, 2, 1 and 0%. On termininating the cultures on day 4, the ratio of 3H-thymidine incorporation between the mixed and the separate cultures was found to be considerably higher in absence of FCS than in the presence of FCS at any of the concentrations indicated. A series of timed cultures, without any FCS, showed a ratio between the incorporation of 3H-thymidine in the mixed cultures and the sum of incorporation in cultures of separate cells which reached a maximum level of 11-15, on day 5 or 6. In tests with cells of other sources, it was found that thymus cells could serve as a source of allogeneic antigen, but could not incorporate thymidine, and that bone marrow cells could be stimulated, but to a lower degree th...

Ultrastructural Analysis of Hemolysin-Forming Cell Clusters

Abstract Following in vitro stimulation of mouse spleen cells with sheep red blood cell antigens (SRBC) many of the resulting hemolysin-forming cells have been found to be associated with cell clusters (1, 2). We have initiated a series of studies designed to investigate the interrelationships of the various cell types found within these clusters. We report here preliminary morphologic observations of hemolysin-forming cell clusters. Dissociated mouse spleen cells were cultured in the presence of SRBC using Saunders' (3) modification of Marbrook's (4) technique. After 48 hr of incubation, the spleen cells were carefully recovered and transferred into 15-ml centrifuge tubes which were then placed in an icebucket. Cell clusters were allowed to settle out in the cold for 10 min. The supernatant containing the remainder of the cells and medium was discarded. The recovered cell clusters were analyzed for hemolytic activity by the method of Jerne and Nordin (5).

Cellular Recognition by Mouse Lymphocytes in Vitro

Abstract The in vitro stimulation of mouse spleen cells by foreign histocompatibility antigens is described. Spleen cells from mice less than 3 weeks of age could not be stimulated in mixed lymphocyte cultures. They were also deficient in their ability to stimulate adult allogeneic cells in mixed lymphocyte cultures. The ability of the cells to be stimulated by histocompatibility antigens was correlated in time with their ability to stimulate allogeneic cells. Stimulation of mouse spleen cells was accomplished by allogeneic spleen cells with differences at the H-2 locus or at the H-3, H-13 loci, and with soluble H-2 antigen. F1 hybrid spleen cells were stimulated by parental strain spleen cells or by parental strain red blood cell stroma. Immunization with allogeneic spleen cells was found to transiently affect the degree of stimulation obtained in mixed lymphocyte cultures of spleen cells from the immunized mice mixed with mitomycin-treated cells of the strain used for immunization. The cells from immunized animals also showed an increased degree of stimulation when reacted in MLC with allogeneic cells sharing some of the H-2 antigens of the strain used for the immunization. Immunization did not affect the degree of stimulation obtained in MLC of cells from strains with weak histocompatibility differences. The possible explanations of in vitro lymphocyte stimulation by histocompatibility antigens of allogeneic or semi-allogeneic cells are discussed.