Induction of the immunologic response in vitro

Immunological response (primary and secondary) was induced in a suspension of mouse splenic cells on nutrient media containing embryonic calf serum or serum against the erythrocytes of an animal--the lymphoid cells donor. The in vitro immunological response was accompanied by a specific increase in a number of the hemolysin-forming and rosette-forming cells. The optimal for induction of the immunological response in vitro was a dose of 10(7) erythrocytes per 1 ml of the culture. It was shown experimentally that antierythrocytic serum could be used instead of the embryonic calf serum to induce the immunological response in vitro. An increase in the count of rosette-forming cells and no increase of the hemolysin-forming cell count was observed on the nutrient media without 2-mercaptoethanol.     

Different effects of the capsular polysaccharide of Klebsiella pneumoniae on the functions of various subpopulations of B cells in the immune response of mice to T-dependent antigen.

Using the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) as a polyclonal B-cell activator (PBA) and sheep red blood cells (SRBC) as a T-dependent antigen, we studied the effects of PBA on the functions of various subpopulations of B cells in the immune response of mice to T-dependent antigen. Antibody-forming cells (AFC) of IgM and IgG types were estimated as anti-SRBC direct and indirect plaque-forming cells (PFC), and the B cells with precursor activities involving generation of AFC and supplementing new B cells as rosette-forming cells (RFC) of the B-cell type. Stimulation of normal mice by CPS-K caused a definite increase in the number of direct PFC but not in that of indirect PFC and RFC in the spleens. The responsiveness of spleen cells of CPS-K-treated mice to generate PFC and RFC responses to a subsequent injection of SRBC was lower than that of CPS-K-untreated normal mice. In this case, the responsiveness to generate RFC and indirect PFC was inhibited more strongly by CPS-K than that to generate direct PFC. When CPS-K was injected into normal mice simultaneously with SRBC, CPS-K never decreased but increased the levels of PFC and RFC responses to SRBC. In the spleens of SRBC-primed mice, the number of RFC was markedly decreased following injection of CPS-K, the number of direct PFC was increased only slightly and the number of indirect PFC was increased very slightly. The responsiveness of spleen cells of these CPS-K-treated SRBC-primed mice to generate secondary PFC and RFC responses to a subsequent injection of SRBC was much lower than that of CPS-K-untreated SRBC-primed mice. In this case, the responsiveness to generate the secondary RFC and indirect PFC responses was more strongly inhibited by CPS-K than that to generate the secondary direct PFC response. When CPS-K was injected into SRBC-primed mice simultaneously with the secondary injection of SRBC, there were marked decreases in the level of the secondary RFC response and slight decreases in that of the secondary indirect PFC response, but little change in that of the secondary direct PFC response. From these results it has been concluded that CPS-K provides the positive signal (the minor action) and the negative signal (the major action) to various subpopulations of B cells functioning at various stages of the immune response to T-dependent antigen in different ways, and acts to regulate the levels of B-cell responses to the antigen-mediated positive signal.     

Influence of a sublethal irradiation on the immune response of the mouse to sheep erythrocytes]

In the CBA mice, the immunological response of the spleen cells (RFC and PFC direct and indirect) against the sheep erythrocytes is highly depressed by a 400 R dose of X rays. The recovery is not complete at the 30th day after irradiation. The response of the bone marrow cells either irradiated or unirradiated to the antigenic stimulation is very low.     

IgM and IgG rosette formation in the primary and secondary immune response in the system of syngeneic transfer of spleen cells]

In the system of syngenous transfer of cells a study was made of the dynamics of IgM- and IgG- of the rosette-froming cells of the spleen in the primary and secondary immune response in mice of the CBA inbred strain immunized with sheep erythrocytes. It was shown that in prolongation of the interval between donor immunization and the transfer of cells to the recipient with his simultaneous immunization for up to 40 days there occurred an increase of the IgG-memory; as to IgM memory-it is expressed with shorter intervals. It is supposed that rosette-forming cells were not bearers of immunological memory.     

Alterations in antigen-induced DNA synthesis by specifically localizing cells and other lymphoid cells as a function of immunological memory

The effect of prior immunological experience on the antigen-induced DNA synthesis by specifically localizing cells (SLC) and other cells capable of migrating into lymph nodes (nonspecific cells, NSC) has been studied. Prior immunization with a homologous red cell antigen (sheep red blood cells, SRBC) results in enhanced DNA synthesis, relative to controls pretreated with saline, by NSC but not SLC for the first 48 hr following secondary immunization. By 72 hr both SLC and NSC show markedly reduced DNA synthesis, the SLC showing the greater reduction. Prior immunization with the strongly cross-reacting antigen, ox red blood cells (ORBC), shows a similar but milder effect; prior immunization with the non-cross-reacting antigen chicken red blood cells (CRBC) is without effect, though a similar depression of DNA synthesis by both SLC and NSC occurs 72 hr after secondary immunization with CRBC. In the presence of a concomitant secondary reaction to a non-cross-reacting (CRBC) or very weakly cross-reacting (horse red blood cells, HRBC) antigen, DNA syntheses by SLC-SRBC and NSC are depressed to approximately the same extent, suggesting that the greater depression of DNA synthesis by SLC seen in homologous secondary reactions reflects the additive effects of clonally specific and nonspecific suppressive factors. A comparison of the DNA synthesis by cross-reacting (ORBC) and non-cross-reacting (SRBC) SLC in mice given SRBC following preimmunization with ORBC suggests that “educated” SLC are considerably more resistant to the effects of nonspecific suppressors than “naive” SLC. Passively administered antibody, in concentrations that markedly reduced the numbers of direct plaque-forming cells appearing 72 hr after primary immunization, had no significant effect on DNA synthesis by SLC or NSC.     

Depression and stimulation of immunologic memory by typhoid lipopolysaccharide and polysaccharide]

The effect of typhoid lipopolysaccharide (LPS) and polysaccharide (PS) on immunological memory in the system of IgM and IgG synthesis and rosette-forming cells was studied. When introduced into animals previously immunized with SRBC, PS stimulated, under certain conditions, immunological memory in the system of IgM synthesis and rosette-forming cells, while the injection of LPS induced only an insignificant stimulation of immunological memory. No stimulation in the system of IgG synthesis was observed after the injection of both LPS and PS. The suppression of immunological memory was noted in the animals receiving LPS as well as in those receiving PS the immunosuppressing effect produced by LPS was more pronounced.     

Modification of immune competence by parasitic infections. I. Responses to mitogens and antigens in mice treated with Trichinella spiralis extract

Spleen cells from mice pretreated with a Trichinella spiralis extract (TsE-mice) showed severe depression of the response to lipopolysaccharide (LPS) and to concanavalin A (Con A), slight depression to phytohemagglutinin (PHA) and normal response to tuberculin purified protein derivative (PPD) as compared to saline-pretreated controls. Mice pretreated with bovine serum albumin (BSA-mice) revealed greatly reduced responses to LPS, somewhat reduced response to Con A, and normal responses to PHA and to PPD. Only TsE-mice showed significant reduction in the number of rosette-forming cells and of direct and indirect plaque-forming cells (DPFC and IPFC). BSA-mice exhibited some reduction of the DPFC only. Direct hemagglutinating (HA) titers were equivalent in the 3 groups after immunization with sheep erythrocytes but facilitated HA titers were depressed in TsE-mice. The total number and the number of viable cells were similar in the spleens of all animals. TsE treatment causes a reduction in the number of T1 lymphocytes and an inhibition of the late differentiation of B cells in the spleen. Suppressor T-cells apparently play a major but not exclusive role in T. spiralis-induced nonspecific immunodepression.     

Effects of cocaine and morphine on IgG production by human peripheral blood lymphocytes in vitro

Elevated serum levels of IgG are amongst the immunological abnormalities exhibited by intravenous drug addicts. We therefore addressed the hypothesis that cocaine and morphine (the major metabolite of heroin) exert a direct effect on human B cell function in vitro. Human peripheral blood mononuclear cells from normal individuals were incubated for 7 days with the T cell-dependent B cell activator pokeweed mitogen (PWM) and serial dilutions of either cocaine or morphine. At the end of this time total IgG was measured by use of a sandwich ELISA incorporating a biotin-labelled affinity-purified anti-IgG and streptavidin peroxidase. At concentrations relevant to those found in plasma, morphine and cocaine did not affect PWM-stimulated IgG synthesis in vitro. We suggest that these drugs of abuse do not directly influence human B cells, but in vivo exert immune modulatory effects via indirect mechanisms.     

Aggregated immunoglobulins as antigen-binding receptors of immune lymphocytes]

At the peak of the primary immune response to sheep erythrocytes there appeared in the spleen of mice rosette-forming cells (RFC) effectively inactivated with antibodies against aggregated mouse immunoglobulins and with the complex of polyadenylic-polyuridylic acids (poly-A, poly-U, respectively). These cells disappeared from the spleen on the 9th day after the primary immunization and were not revealed at the peak of the secondary immune response. When small splenic lymphocytes obtained on the 5th day after the immunization with sheep erythrocytes were incubated in vitro for 24 hours the total amount of the RFC inactivated by antibodies to the aggregated mouse immunoglobulin disappeared completely. These data can be considered as an indication of the existence at the peak of the primary immune response of rosette-forming cells having the antigen-antibody complexes in the capacity of the antigen-binding receptors.     

Effect of a typhoid lipopolysaccharide on the primary and secondary immunological response to ram erythrocytes

The effect of typhoid bacterial lipopolysaccharide (LPS) on the primary and secondary response to sheep red blood cells was studied. LPS, injected simultaneously with the antigen, stimulated the synthesis of IgM and IgG, as well as the production of rosette-forming cells. When injected on days 2 and 3 after the secondary immunization, LPS induced the maximum stimulation of IgM, IgG and rosette-forming cells, while the injection of LPS prior to immunization induced immunosuppression which particularly affected IgG and rosette-forming cells.     

Preparation of mouse antiserum against isologous aggregated immunoglobulins and its effect on rosette forming cells]

A method of obtaining antisera against isologous aggregated mouse immunoglobulins is described. This serum designated MAAS blocked in vitro the antigen-binding receptors of the immune rosette-forming cells. MAAS was injected to mice immunized with SRBC. In comparision with the immunized mice given normal isologous serum rosette-forming B-cells were absent in the spleen of mice given MAAS at the peak of isologous response. But the antibody-forming cell count was not decreased under the influence of MAAS.     

Induction by an immunogenic immunomodulating agent of nonspecific T cell suppression of lymphocyte responsiveness in MLR but not of antibody production

Summary Spleen cells derived from BALB/c mice that had been repeatedly immunized with the methanol extraction residue (MER) fraction of tubercle bacilli exhibited a depressed capacity to act as responder cells in allogeneic and syngeneic mixed lymphocyte reactions (MLR). Previously reported studies revealed that such spleen cells are also defective in the in vitro generation of antibodies. In order to determine the nature of the cells responsible for the depressed MLR reactivity, purified populations of splenic macrophages, B lymphocytes, T lymphocytes originating from normal and from MER-immunized mice, and cell culture supernatants were added to MLR mixtures consisting of normal mouse splenocytes. Macrophages originating from MER-immunized mice and their culture supernatants exerted a significantly higher suppressive effect on MLR than that of corresponding preparation from normal mice. Splenic T cells originating from MER-immunized mice and their supernatants also significantly suppressed the MLR response. However, the same T cell populations that were inhibitory in MLR failed to suppress the in vitro generation of antibodies against sheep red blood cells in the presence of either MER or 2-mercaptoethanol. These and previously reported findings indicate that a nonspecific immunomodulating agent, MER, can, under certain conditions of treatment, elicit the induction of nonspecific suppressor T cells for MLR but not for antibody production, and, accordingly, can inhibit cellular and humoral immunological responsiveness by different mechanisms.     

Structural and serological studies of lipopolysaccharides of Citrobacter O35 and O38 antigenically related to Salmonella

Abstract Lipopolysaccharide (LPS) was administered into sheep red blood cells (SRBC)-primed mice, and the effect of LPS on SRBC-specific memory cells was investigated. Spleen cells from SRBC-primed mice which were injected with LPS exhibited much lower in vitro secondary plaque-forming cells (PFC) responses to SRBC than those from untreated SRBC-primed mice. The in vitro anti-SRBC response of the spleen cells to LPS was also reduced. The combination experiments of B cells and T cells from SRBC-primed mice which were injected with or without LPS demonstrated that the reduction of immune responses to SRBC after administration of LPS was caused by the defect of SRBC-specific B memory cells, but not T memory cells. B cell type rosette-forming cells (RFC) for SRBC markedly decreased after injection of LPS, while PFC as antibody-forming cells did not increase subsequently. Therefore, the reduction of RFC was not due to their differentiation into PFC. The lymphoid follicles in the spleens from mice injected with LPS were stained positively by in situ nick end labeling specific for fragmented DNA. A large percentage of Ig⁺ spleen cells from SRBC-primed mice which were injected with LPS was also stained positively. The injection of glucocorticoids into SRBC-primed mice induced similar reduction of B memory cells. It was suggested that LPS might induce apoptosis of B memory cells and regulate B cell memory in antigen-nonspecific manner.     

Effects of intravenous silica on immune and non-immune functions of the murine host.

Silica, an agent toxic for macrophages, administered i.v. to DBA/2 mice rapidly depresses the clearance of colloidal carbon by the reticuloendothelial system and reduces the in vitro phagocytic activity of peritoneal macrophages harvested 3 days after silica injection. Silica blocks the humoral immune response to sheep erythrocytes and the cell-mediated immune response to allogeneic fibroblasts when given before antigen. Silica also induces complex alterations in spleen cell responsiveness to concanavalin A involving both local and serum factors. Silica had no significant effect on the induction of interferon by statolon or Newcastle disease virus. No unequivocal evidence was obtained that silica has a direct depressive effect on cells other that macrophages, but indirect effects on lymphocytes were produced most likely by factors released from silica-lysed macrophages. Intravenous silica may prove useful for the separation of interferon induction and immune response stimulation in studies of host resistance to infection and oncogenesis. Considerable variation exists in the immunodepressive effects of different preparations of silica.     

Multiple myeloma: an immunologic profile. I. Peripheral blood studies.

Seventy-four patients with multiple myeloma, 17 untreated and 57 treated, were studied to characterize their peripheral blood lymphocytes. PBL were studied for E, EAC, and EA rosette-forming cells, SIg, and Fc receptor-bearing cells. The responses to HA, Con A, and PWM were assessed as well as their ability to stimulate or to respond in a MLC. Finally, the capacity of mitogen-stimulated lymphocytes to lyse Chang cells, CRBC, and PHA-stimulated lymphoblasts was examined. These results were compared with a group of normals and patients with benign monoclonal gammopathy. In untreated myeloma patients there was a normal percentage of T cells, but an abnormal distribution of B cells as judged by a decrease in SIg-bearing cells, as well as an increase in EAC rosette-forming cells. Subpopulation analysis showed a marked increase in EAC rosette-forming cells without SIg. PHA, Con A, and PWM, and response in MLC were all normal. However, the ability to stimulate in MLC was significantly depressed. Treated myeloma patients had similar findings, except that the response to PWM was significantly depressed. The capacity of PWM-stimulated cells to lyse target cells was depressed in both groups. The results indicate that, in the peripheral blood of myeloma patients, there are populations of lymphocytes characterized by the presence of the EAC receptor without SIg, which are deficient in the capacity to stimulate an MLC response and the ability to be cytotoxic when stimulated by PWM. The results form a baseline for the study of abnormal lymphoid function in human myeloma.     

Distinct response of human B cell subpopulations in recognition of an innate immune signal,CpG DNA

Innate immunity has recently gained renewed interest in its ability to regulate adaptive immunity. Among the innate immune signals, CpG DNA has revealed its potential as a vaccine adjuvant. However, the cellular mechanism for the effect of CpG DNA on the humoral immune response is not well understood. Here, we investigated the effects of CpG DNA on human B cell differentiation using highly purified B cell subsets: naive, germinal center (GC), and memory B cells. In the in vitro culture system that mimics the primary or secondary immune response in vivo, CpG DNA markedly augmented the proliferation and generation of plasma cells from naive and memory B cells. CpG DNA dramatically increased plasma cell generation from GC B cells. However, CpG DNA did not have effect on memory B cell generation from GC B cells. These results suggest that CpG DNA potentiates the B cell adaptive immune response by enhancing terminal differentiation, but does not affect the generation of memory B cells.     

Liver parenchymal cell proliferation during secondary induction with estradiol-17 beta in Xenopus

We have previously demonstrated that injection of adult male frogs with estradiol-17 beta causes extensive proliferation of liver parenchymal cells together with the induction of vitellogenin (R. J. Spolski, W. Schneider, and L. J. Wangh (1985) Dev. Biol. 108, 332-340). In addition, purified parenchymal cells placed in culture synthesize DNA in an estrogen-dependent manner (B. S. Aprison, L. Martin-Morris, R. J. Spolski, and L. J. Wangh (1986) In Vitro 22, 457-464). We now describe conditions under which secondary exposure to estradiol-17 beta, either in vivo or in vitro, can lead to further DNA synthesis and cell division. The extent of this proliferation depends upon both the magnitude of the primary dose and the length of time elapse before secondary stimulation. A hormone dose of 0.5 mg, which causes little cell proliferation initially, allows for maximal secondary proliferation in response to 2.0 mg, while a maximal primary dose of 2.0 mg substantially inhibits further division in response to a secondary treatment with the same hormone dose. Cell culture experiments demonstrate that the failure of liver cells, in maximally stimulated males, to synthesize DNA in response to estrogen is not irreversible. But, cell crowding in culture does restrict DNA synthesis. The restrictions seen in vivo may therefore be due to structural features of the intact tissue rather than to terminal differentiation at the genetic level. These results are discussed with regard to our understanding of hormone-dependent differentiation in the frog liver system.     

Rosette formation and humoral immune response to 2,4-dinitrophenol in mice of different inbred strains]

A study was made of the content of rosette-forming cells to DNP-ovalbumin in the spleen of mice of different inbred strains. The values of the rosette-forming cells and of the titre of serum agglutinins to the DNP-group in DNP--bovine gamma-globulin immunization of mice of these strains were determined. It was shown that there were interstrain differences in respect to the normal and immune rosette-forming cells and also humoral antibodies. There was noted a direct correlation between the number of the immune rosette-forming cells and the antibody titre in the serum of mice of the same inbred strain. Immune response (both by the content of rosettes in the spleen and by the antibody level) proved to be the minimal in mice with the highest level of rosette-forming cells. There proved to be no inverse relationship.