Effect of antiserum against isologous aggregated immunoglobulins on the delayed-type hypersensitivity in mice immunized with sheep red blood cells

The mouse antiserum against isologous aggregated immunoglobulins (MAAS) injected to mice sensitized with 10(5) sheep red blood cells (SRBC) did not influence the delayed-type hypersensitivity (DTH) tested on the peak of sensitization (the 4th day) but enhanced significantly DTH tested on the 6th day. MAAS completely abolished the DTH suppression observed after sensitization with 5 x 10(7) SRBC. In transfer experiments the number of the DTH suppressor cells decreased in the spleen of sensitized mice under the MAAS action. MAAS did not affect the proliferation of antibody-forming cells (AFC) and hemagglutinin production but reduced by 70% the number of rosette-forming cells (RFC) in the spleen on the peak of the initial immune response. The data obtained may indicate that RFC participate in DTH suppression.     

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.     

Structural organization of the antigen-binding receptors of immunocompetent cells]

It was demonstrated in this work that rabbit antimouse serum against the aggregated immunoglobulins (RAAS) and mouse serum against the aggregated mouse immunoglobulins (MAAS) inhibited the rosette-forming B-cells (RFC) on the 5th day after the immunization of mice CBA with SRBC in a dose of 5 X 10(-8) cells in vitro in 1:20--1:80, and 1:10--1:40 dilutions in 83--55 and 72--39%, respectively. In difference from RAAS, MAAS in a dilution of 1:20 induced a statistically significant suppression of the antigen-binding receptors of RFC of-B type in the intact animals, and on the 8th--9th day after their immunization with SRBC. In vivo MAAS induced inactivation of the antigen-binding receptors of B-lymphocytes only. Results of the work carried out served as a confirmation of the fact that immunoglobulins in the form of an antigen-antibody complex (functioning in the capacity of the antigen-binding receptors) were sorbed on B-lymphocytes of the spleen.     

Antigen-dependent induction of a non-specific humoral factor blocking rosette-forming cells in experiments in vitro and in vivo

Isologous serum of CBA mice immunized with rabbit immunoglobulins (ARIS) contained a factor capable of inactivating rosette-forming splenocytes (RFC) in vitro from the same strain of mice immunized with SRBC. When mouse SRBC immunization was carried out against the background of ARIS injection the court of RFC to SRBC at the peak of immune response was 30% of that of mice injected with SRBC and normal isologous serum. A decrease of RFC count was the result of disappearance of the theta-negative RFC. Passive ARIS immunization failed to influence the antigen-induced proliferation of the antibody-forming cells and the synthesis of antibodies against SRBC.     

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.     

Selective concurrent suppression of the process of rosette-forming cell accumulation during the immune response]

Repeated injections to mice of normal rabbit immunoglobulins preceding immunization with sheep erythrocytes inhibited the accumulation of rosette-forming cells (RFC) in the spleen, without influencing the proliferation of the antibody-forming cells and hemaggutinin production. Reduction of the RFC under these conditions occurred on account of B-cells whose antigen-binding receptors could be blocked by antibodies against the aggregated mouse immunoglobulins and a complex of polyadenylic-polyuridylic acids. Repeated injections of the competitive antigen enhanced the formation of the immunological memory to the second antigen. The problem of the origin of the immune rosette-forming B-cells and their influence on the formation on the immunological memory is discussed.     

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 sera against aggregated mouse immunoglobulins on a population of lymphoid and hematopoietic cells]

The in vitro treatment of the mouse spleen cells immunized by the ram erythrocytes with the rabbit and mouse sera against the thermoaggregated mouse immunoglobulins resulted in the inhibition of antigen binding receptors of rosette forming cells. The mouse serum, unlike the rabbit one, induced the inactivation of receptors in rosette forming lymphocytes both in the non-immune and immune mice on the 8th day after the antigenic stimulation. The treatment of bone marrow cells from the intact mice with these sera increased insignificantly the number of hemopoietic colonies in the spleens of lethally irradiated syngenic recipients and stimulated markedly the migration of spleen cells. This may be due both to the direct effect of these sera and to their mediated (through the humoral factor) influence. The inactivation of antigen binding receptors in the spleen rosette forming cells suggests the presence of immunoglobulins on the membrane of B-lymphocytes in the aggregated state or in the form of antigen--antibody complexes.     

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.     

Types of cells involved in antigen-stimulated and spontaneous rosette formation with Toxoplasma gondii.

Antigen-binding cells were identified by using rosette formation of Toxoplasma gondii and defined lymphoid populations under different experimental conditions. Treatment of immunized spleen cell suspensions with anti-Thy 1 serum plus complement inhibited 5 to 29% of the rosette-forming cells (RFC). Higher numbers of thymus-derived lymphocyte-RFC were obtained after incubation at 4 degrees C and by the centrifugation method than by simple incubation at 20 degrees C. RFC were also observed with thymocytes. Combined treatment with anti-Thy 1 serum plus complement and depletion of adherent cells indicated that the major proportion, 46 to 70%, of RFC were B cells. Spleenocytes of nu/nu mice formed similarly high numbers of rosettes. Spontaneous RFC were observed in nonimmunized mice with both spleen and thymus populations. Numbers of rosettes varied considerably depending on the method and the source of cell population used. Removal of adherent cells from spleen suspensions resulted in RFC reduction of 14 to 25% in immunized and 14 to 33% in nonimmunized animals. Pretreatment with anti-mouse immunoglobulin inhibited completely the spleen and spontaneous thymus RFC and partially the thymus RFC in immunized animals.     

Antibody formation in mouse bone marrow. VIII. Dependence on potentially circulating memory cells: a study with parabiotic mice.

Mouse bone marrow barely contains antibody-producing plaque-forming cells (PFC) during the primary response to sheep red blood cells (SRBC). However, during the secondary response, the number of IgM, IgG, and IgA PFC in the bone marrow can rise to a level which surpasses the number of PFC in all the other lymphoid organs together. In the present paper we investigated whether the capacity of immune mice to react upon a booster injection of SRBC with a bone marrow PFC response can be transferred from immune to nonimmune mice. Therefore, mice primed with SRBC 6 months previously and nonprimed syngeneic mice were joined for parabiosis and were separated from each other at various intervals after joining. These separated mice were subsequently immunized with SRBC. It was found that, after 3 weeks of parabiosis, the nonprimed members reacted upon an injection of SRBC with a bone marrow IgM, IgG, and IgA PFC response as high as did the previously primed members. Furthermore it could be demonstrated by means of cell transfer experiments that, after a period of parabiosis of 3 weeks, the bone marrow and spleen of the normal mice contained about as many memory cells as the bone marrow and spleen of the immune mice. These results suggest that antibody formation in mouse bone marrow is dependent on a population of potentially circulating memory cells.     

Analysis of certain mechanisms of antigen-specific suppression of the immune response

CBA mice were immunized with sheep red blood cells (SRBC) to obtain immune spleen cells (ISc) which were used to suppressor cells. Administration of ISC to intact syngeneic recipients on the immunization day led to a more powerful suppression of the immune response as compared to that seen one day after antigen injection. Four days after immunization the animals' immune response was not liable to be suppressed. ISC extract possessed similar effects with respect to the immune response of normal spleen cells which were transplanted to the cyclophosphamide-treated recipients. The immune response of spleen cells from mice immunized with SRBC in a dose of 10(6) was less liable to be suppressed. Hyperimmune spleen cells from donors immunized with SRC in a dose of 10(9) were insensitive to ISC or to the extract. Experiments with the use of adoptive transfer of a mixture of immune and intact T- and B-cells have disclosed that B-cells from hyperimmune donors were resistant to suppression. Therefore, B-lymphocytes are the most probable target cells exposed to T-suppressors in the given system. The mechanism is discussed of the selective effect of T-suppressors on B-cells in the course of the immune response development during immunization with high doses of antigen.     

Antigen-reactive cell opsonization: a mechanism of antibody-mediated immune suppression.

Antisera against sheep red blood cells (SRBC) specifically suppressed the direct anti-SRBC plaque-forming cell (PFC) response in mice when passively administered with the antigen. The suppressive activity of mouse and rabbit anti-SRBC sera was found to correlate with anti-SRBC opsonic activity but not with hemagglutination or hemolysin titers. Macrophage depletion of mice, using carrageenan treatment, inhibited antibody-mediated immune suppression. When mice immunized with SRBC were given ¹²⁵I-labeled Udr, radiolabeled spleen lymphocytes were obtained which specifically formed rosettes with SRBC. These radiolabeled antigen-reactive cells (1ARC) were specifically opsonized in mice treated with antigen-antibody complexes but not in mice treated with antigen or antibody alone. These results suggest that antibody-mediated immune suppression may be due to specific opsonization (and subsequent destruction) of ARC in the presence of antigen-antibody complexes.     

B-cell suppression of antibody response to sheep red blood cells in mice of high- and low-responding genotypes.

CBA and C57B1 mice (high and low responders to sheep red blood cells, respectively) were injected intravenously with syngeneic lymph node, marrow, spleen, or thymus cells together with sheep red blood cells (SRBC), and the production of antibody-forming cells (AFC) was assayed in the spleen. Transfer of lymph node, marrow, spleen, or thymus cells led to a significant enhancement of immune responsiveness in low-responding C57B1 mice. In contrast, transfer of marrow, lymph node, or spleen cells to high-responding CBA mice was accompanied by a decline in AFC production. These effects were magnified if syngeneic cell donors had been primed with SRBC; suppression in CBA mice and stimulation in C57B1 mice were especially pronounced after transfer of SRBC-primed lymphoid cells. Pretreatment of CBA donors with cyclophosphamide in a dose causing selective B-cell depletion completely abrogated the suppression of immune responsiveness. A large dose (10⁷) of syngeneic B cells injected together with SRBC suppressed the accumulation of AFC in both CBA and C57B1 mice. No suppression of immune responsiveness was observed after transfer of intact thymus cells, hydrocortisone-resistant thymocytes, or activated T cells. We conclude that suppression of the immune response to SRBC is induced by B cells. At the same time, there is a possibility that the addition of “excess” B cells acts as a signal, triggering suppressor T cells.     

Mechanisms of action of synthetic polyelectrolytes and polyampholytes on the immune response]

We investigated the effect of polyacrilic acid (PAA) on the immune response in mice of various strains on sheep red blood cells and also the influence of poly-2-methyl-5-vinyl-pyridine (PMVY), PAA and their statistical copolymers on antibody-forming cells (AFC) production in cultures of T- and B-lymphocytes in vivo. PAA was seen to increase accumulation of AFC in the spleen of mice depending on their genotypes. PMVP and PAA were found to intensify the cooperating interaction of T- and B-lymphocytes, whereas their copolymers exert quite an opposite effect. The injection of copolymers to the recipients of cooperating T- and B-lymphocytes practically results in the complete elimination of the cooperation effect between T- and B-lymphocytes in the immune response to sheep erythrocytes without cytostatic action of cell proliferation.     

Resistance to Naegleria fowleri infection passively acquired from immunized splenocyte, serum or milk

A pathogenic free-living amoeba, Naegleria fowleri, causes primary amoebic meningoencephalitis to human and experimental animals. This infection is rare, but the mortality is very high. Nowadays, drug treatment or active immunization of human or mice are being tried with partial effectiveness. This study shows passive immunization effect by transfer of immunized spleen cells, serum, or milk from immunized mother in mouse experimental model. Young BALB/c mice were immunized intraperitoneally with 2-3 X 10(6) trophozoites of N. fowleri, and spleen cells and sera were collected for injection to recipient mice. There were seven transfer groups, i.e., immunized mouse serum, spleen cells, serum and spleen cells, normal mouse serum, spleen cells, serum and spleen cells, and control group. Three days later, BALB/c mice were inoculated with 1 x 10(4) trophozoites of N. fowleri intranasally. After infection, decreased mortality and prolonged survival time of mice were noted in immunized groups compared with non-immunized control group. The groups injected with immunized spleen cells or normal serum showed lower mortality than that of controls but showed no changes of serum IgG level. The groups injected with immunized serum or normal spleen cells showed increased serum IgG level after immunization but hundred percent mortality was observed. Mother mice were immunized intraperitoneally with 2-3 X 10(6) trophozoites of N. fowleri at the end of pregnancy and weaning period. Soon after the delivery, litters born of non-immunized mother were matched with immunized mother for feeding immune milk. After three weeks, the litters were infected with 1 X 10(4) trophozoites of N. fowleri or sacrificed for serum collection to measure the IgG levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunosuppressive effect of Entamoeba histolytica extract on hamsters

The immune response to sheep red blood cells (SRBC) in mice and hamsters injected with an extract of entamoeba histolytica was studied. Both the primary and secondary immune response, measured by anti-SRBC antibody titers, were unaltered in the mouse, while a significant depression of the primary, but not the secondary, response was observed in the hamster. The effect was greatest when the amebic extract (AE) and SRBC were injected on the same day. The number of anti-SRBC rosettes formed in the spleen cells of hamsters treated with both AE and SRBC on day 0 was measured from days 1-16. The response peaked on day 13, while cells from animals injected with SRBC alone gave a maximal response on day 5. The formation of anti-SRBC rosettes in T-lymphocyte-enriched spleen cells treated with anti-gamma globulin serum and complement was almost abolished for the duration of the experiment. It is suggested that the mechanism responsible for this immunosuppressive phenomenon could involve early interference in the afferent limb of the immune response.     

Study of the phenomenon of specific suppression of the immune response in an adoptive transfer system]

It was revealed that the administration of the spleen cells (SC) of syngeneic animals immunized with a high dose of sheep red blood cells (SRBC) to intact mice led to a marked specific suppression of the recipients' immune response. The donors' SC obtained on the 14th day after the intraperitoneal injection of SRBC had the greatest suppressive activity. The SC of intact animals and mice given rat erythrocytes preliminarily failed to influence the immune response of the intact recipients in their SRBC immunization. Treatment of immune SC with the anti-T-serum (ATS) or the anti-B-globulin (ABG) and the complement considerably decreased or completely eliminated the suppressive activity. Administration of a mixture of two immune SC suspensions one of which was ATS- and another ABC-treated did not produce any suppression of the immune response in the intact recipients. It is supposed that the suppressor cells in the given model were T-lymphocytes expressing the antigens, common of cross-reacting with the B-cells.     

Enhanced antibody response in retrovirus-infected mice treated with endotoxin or nontoxic polysaccharide derivative

Friend leukemia virus (FLV) is a retrovirus which causes marked suppression of the immune response of genetically susceptible mice. In the present study the depressed antibody response to sheep erythrocytes by spleen cells from FLV-infected mice was partially reversed by injection of either a bacterial endotoxin or a nontoxic polysaccharide derivative directly into infected mice or by addition to spleen cell cultures from these mice immunized in vitro with sheep red blood cells (SRBC). The endotoxin and PS in a dose-related manner markedly increased the antibody responsiveness of the spleen cells to SRBC. Thus these results indicate that the nontoxic polysaccharide derivative has properties equivalent to the toxic endotoxin in enhancing the antibody responsiveness of FLV-suppressed spleen cells to a T-cell-dependent antigen like SRBC.     

Regulation of B-lymphocyte production in the bone marrow: mediation of the effects of exogenous stimulants by adoptively transferred spleen cells

The cellular mechanism by which an injection of sheep red blood cells (SRBC) results in an increased production of B lymphocytes in mouse bone marrow has been studied by adoptive cell transfer and the use of two in vivo assays of bone marrow B-cell genesis. Proliferation of B progenitor cells was examined by immunofluorescent labeling combined with mitotic arrest, while small lymphocyte renewal was measured by [3H]thymidine labeling and radioautography. In C3H/HeJ mice the administration of SRBC resulted in increased proliferation among bone marrow pre-B cells which contained cytoplasmic mu heavy chains but lacked kappa light chains and surface mu chains. The turnover of small lymphocytes also increased. These stimulatory effects were transferred to naive recipient mice by organ fragments and by cell suspensions harvested from the spleens of donor mice injected with SRBC. In contrast, spleen cells and thymus cells from saline-injected donors and thymus cells from SRBC-injected donors had no such stimulatory effects. The results demonstrate that spleen cells mediate the stimulatory effect of SRBC on bone marrow B-lymphocyte production. Spleen cell transfer provides a system to study further the cells and factors involved in the regulation by external environmental agents of the rate of primary B-cell genesis in vivo.