Suppression Of Antibody Formation Research Articles

Cholera Toxin, Ganglioside Receptors and the Immune Response

Cholera toxin activates plasma membrane adenylate cyclase in all mammalian cell types. The structure-function relationship of the toxin has recently been clarified, and the cell membrane receptor identified. This information has made cholera toxin the "agent of choice" for studies in many biological systems of the possible regulatory role of adenylate cyclase/cyclic AMP. This article describes briefly our current knowledge about the toxin and its receptor. It then reviews recent research which has revealed that cholera toxin has strong modulating influences on the proliferation of normal and malignant lympocytes as well as on the initiation and expression of immune responses. The toxin has been found to inhibit DNA synthesis of B and T lymphocytes in vitro without inducing cell death and also to inhibit seems to decrease antibody secretion from plasma cells in vitro, and might also interfere with the release of other soluble immunological mediator subtances. In vivo cholera toxin induces a transient involution of the spleen and a more prolonged lymphocyte depletion of the thymus in mice; these effects appear to be mediated through the adrenal glands. The toxin inhibitors allograft rejection, and either stimulates or suppresses antibody formation depending on the timing of the toxin in relation to the antigen administration. It increases the capacity of the spleen cells to induce graft-vs-host reactions and the "allogeneic effect" on antibody production. An inhibitory effect on a normal suppressor population among the spleen cells has been identified. The findings illustrate the complex effects induced on the immune system by the probably most discriminative investigative tool available for stimulation of the adenylate cyclase/cyclic AMP system.

B cell tolerance induced by polymeric antigens. III. Dissociation of antibody formation and memory generation in tolerant mice

Hapten (2,4-dinitrophenyl (DNP))-substituted type 3 pneumococcal polysaccharide (DNP-lys-S3) effectively suppresses both primary and secondary anti-DNP antibody responses to DNP-proteins. The present experiments show that tolerizing doses of DNP-lys-S3 have much less effect on B cell memory (re)generation in three distinct situations: (i) in serial transfers of DNP-protein-primed cells, (ii) in virgin mice tolerized before priming with DNP-hemocyanin, and (iii) in tolerized, hemocyanin-primed mice boosted with DNP-hemocyanin. Under some conditions tolerized mice developed 10-50% of normal memory in the absence of significant antibody formation. Isoelectric focusing analyses revealed that many B cell clones proliferate in tolerant mice, but produce very little antibody until transferred to further hosts. Clones that escape suppression in partially tolerant mice do not appear to be resistant to DNP-lys-S3, when retested. Since there is independent evidence that the generation of B memory cells is less T cell-dependent than the development of antibody-forming cells, these data are best explained by assuming that DNP-lys-S3 blocks lymphocyte cooperation. This would be expected to preferentially suppress antibody formation, and to spare memory generation.

Localization of brain-associated hematopoietic antigens in the neuronal fraction of brain

Brain shares antigens with several of the cell types of the hematopoietic system. We have demonstrated antibodies in anti-mouse brain antisera which react with thymus-derived lymphocytes (1, 2) hematopoietic stem cells (3, 4) erythrocytes (5), and more recently a subpopulation of peripheral B lymphocytes (Golub, manuscript in preparation). The experiments in this report were designed to localize these antigens to determine if they reside in the neuronal or the neuroglial portions of the brain. Our approach was to fractionate mouse brain into myelin-enriched (glial) and myelin-depleted (neuronal) fractions for which immunological distinctiveness has already been demonstrated (6, 7) and absorb anti-brain antiserum with these fractions. The absorbed antisera were then tested for ability to kill thymocytes, prevent hematopoietic stem cell colony formation, agglutinate erythrocytes, and inhibit antibody formation. It was found that the antigens responsible for all of these activities reside in the neuronal (myelin-depleted) fraction.

Granulomatous Hypersensitivity to Schistosoma Mansoni Eggs in Thymectomized and Bursectomized Chickens

Abstract Early studies in which immunoglobulin was found in the Schistosoma mansoni egg granuloma led to the assumption that antibody played a major etiologic role in the development of this lesion (1, 2). Subsequent investigations, however, revealed that granulomatous hypersensitivity to S. mansoni eggs could be passively transferred with cells and not with serum (3). Further studies confirmed the latter findings in that: 1) complete granulomas consisting largely of macrophages, eosinophils, and lymphocytes form in the absence of any detectable IgG1 or IgG2 antibodies (4, 5); 2) the onset of granuloma formation correlates with the development of delayed dermal reactivity and in vitro correlates of delayed hypersensitivity (4, 5); and 3) suppression of cell-mediated reactivity markedly diminishes granuloma formation (6–8) whereas suppression of antibody formation via x-irradiation (9) or Friend virus leukemia (8) has no effect. In spite of considerable evidence suggesting that the immunologic etiology of the S. mansoni granuloma is virtually wholly cell-mediated, claims for an important role for antibody have continued (10).

Influence of parental lymphoid cells on the production of 19S and 7S antibodies in F1 hybrid mice.

Parental spleen cells injected into F1 hybrid mice stimulated the 7S and, to a lesser degree, 19S primary antibody response to thymus-dependent and -independent antigens. The stimulatory effect on 7S antibody formation as compared to mice, injected with syngeneic spleen cells, was exerted by parental spleen cells injected after, as well as prior to antigenic stimulation. Large amounts of parental spleen cells inhibited the antibody response in comparison to the response obtained after injection of syngeneic cells. Allogeneic cells augmented the 7S and 19S antibody response to very small amounts of antigen. The effect of parental cells on host antibody formation could be abolished by treating the cells with anti-Thy 1 serum and complement which confirms that the stimulatory effect was mediated by T cells. The results indicate that 7S antibody formation is more dependent on non-antigen-specific influences from T cells than is 19S antibody production and are compatible with the hypothesis that activated T cells release substances which stimulate or inhibit antibody formation. These substances do not appear to be responsible for the immunologically specific ‘thymic helper’ effect.

The Effect of Metabolic Inhibitors and Hydroxylamine on the Immune Response in Mice to Mycobacterial Ribonucleic Acid Vaccines

Abstract Metabolic inhibitors were used in an attempt to determine the manner by which mycobacterial ribonucleic acid (myc. RNA) immunizes against tuberculosis. Inhibitors of protein synthesis (streptomycin sulfate, chloramphenicol, cycloheximide) and inhibitors of DNA-dependent RNA polymerase (rifampin) had no effect on the immune response of mice vaccinated with myc. RNA. All of these compounds except streptomycin sulfate, however, inhibited antibody formation under the same conditions. In contrast, inhibitors of deoxyribonucleic acid (DNA) synthesis significantly reduced the immune response. Five-fluoro-deoxyuridine (FUDR), which inhibits thymidine synthetase, when injected just before vaccination, reduced the immune response but had no effect if injected 18 hr after vaccination. These results indicated that early synthesis of DNA was important, and that DNA once formed was not affected by FUDR. Ethidium bromide, proflavine and chloroquine were each mixed with myc. RNA and injected into mice. Under these circumstances the immune response was significantly lowered. In addition, chloroquine was injected at various times and significant reduction in the immune response occurred only if it were injected 1 hr or later after vaccination. This compound binds with DNA, and shows again the necessity for the presence of DNA in the immune process. Actinomycin D injected at the time of vaccination or 1 hr after vaccination had no effect on the immune response. However, if actinomycin D was injected 18 hr after vaccination or injected daily for 4 days after vaccination, a marked reduction in the immune response occurred. These results indicated that actinomycin D, which inhibits RNA transcription may have inhibited the replication of myc. RNA. A mutogenic agent, hydroxylamine, also reduced significantly the immunogenic activity of myc. RNA. These results closely paralleled those obtained by other investigators in studies aimed at eliciting the mode of replication of oncogenic viruses. The replication of these RNA tumor viruses is unique in that DNA synthesis is required for viral RNA replication early after infection, and there is a requirement for RNA transcription. The similarity between the manner by which myc. RNA immunizes and RNA tumor viruses replicate is discussed, and a comparison also is made of the two diseases, tuberculosis and tumors formed by oncogenic viruses. These results strongly suggest that myc. RNA may immunize against tuberculosis by forming a DNA template in the host cell which then transcribes specific information for the replication of myc. RNA, and thereby transforms a normal cell into a specific immune cell. If correct, this would provide a new concept of the nature of cellular immunity of tuberculous infection.

Immunoregulatory α-Globulin: Failure to Inhibit Antibody Response When Administered after Antigen Exposure

Abstract There exists in the serum α-globulins an immunosuppressive factor(s) which was first described by Kamrin (1), and subsequently separated chromatographically by Mowbray (2), and by our laboratories (3, 4). This factor has been demonstrated to prolong skin grafts (3) and to inhibit antibody formation (5, 6). We have also found that this factor inhibits lymphocyte proliferation induced in vitro by both mitogens and antigens (4). We have recently found that the active factor is a polypeptide(s) which migrates electrophoretically with, and may be bound to, an α-globulin protein(s) (7). We have called the active material “immunoregulatory α-globulin” (IRA).4 The mechanism of action of this immunosuppressive factor remains to be determined. Studying mitogen stimulation of lymphocytes in tissue culture, we have observed that the IRA is not suppressive if this agent is added after the lymphocytes have become activated (8). This observation suggests that the IRA acts upon the early events involved in antigen recognition.

Antigen recognition and localization in RES blockaded mice: morphologic and immunologic studies.

Cells of the reticuloendothelial system (RES) obviously constitute the major cell types involved in immunologic events dealing with the maintenance of the body’s integrity against extrinsic and intrinsic parasites, such as microbial pathogens and neoplastic cells. Many recent morphologic and functional studies have helped elucidate some of the complex interactions among the various cells of the RES in immunologic reactions (1–3). In this regard, RES blockading agents have been utilized in attempts to “dissect” some of the cellular steps in the immune response. Studies from this laboratory, as well as from others, have indicated that RES blockade with various particulate substances, including colloidal carbon, induces a marked inhibition of antibody formation in mice (4–8). Pretreatment of mice with colloidal carbon before challenge immunization with an antigen such as sheep erythrocytes generally results in a 90–95 percent or greater suppression in appearance of antibody, as assessed at both the humoral and cellular levels (6–8). Various factors influencing such immunosuppression have been studied in detail, including some of the cell types involved (9).

THE EFFECTS OF MERCAPTOETHANOL AND OF PERITONEAL MACROPHAGES ON THE ANTIBODY-FORMING CAPACITY OF NONADHERENT MOUSE SPLEEN CELLS IN VITRO

Nonadherent mouse spleen cells exhibited poor viability and little or no capacity to form antibodies to sheep red cells in the Mishell-Dutton culture system. Viability and antibody-forming capacity could be restored by addition to these cultures of low concentrations of mercaptoethanol (10(-4)-10(-5)M), or by addition of appropriate numbers of mouse peritoneal macrophages. Macrophage concentrations lower than optimal resulted in lower lymphoid cell viability and correspondingly fewer plaque-forming cells, whereas excess macrophages resulted in marked inhibition of antibody formation despite good viability of the lymphocytes. Restoration of the nonadherent cells with mercaptoethanol was thus much simpler and more reproducible than it was with macrophages; furthermore, the number of plaque-forming cells developed in cultures restored with mercaptoethanol was approximately fourfold higher than it was in cultures restored with optimal numbers of macrophages. In the presence of mercaptoethanol, the plaque-forming capacity of the nonadherent spleen cells was not increased when small numbers of macrophages were added to the system, nor was it decreased when the few macrophages present in the nonadherent cells were further reduced or eliminated. Excess macrophages inhibited antibody formation in the cultures containing mercaptoethanol as they did in control cultures. Optimal restoration of plaque-forming capacity to the nonadherent spleen cells with mercaptoethanol required the reducing agent to be present throughout the 4 or 5 day culture period. Addition of mercaptoethanol 1 or more days after initiation of culture, or transfer of the cells to a medium free of mercaptoethanol before completion of the culture resulted in a reduction in the numbers of plaque-forming cells. The results suggest that mouse lymphoid cells do not require macrophages in order to form antibodies to sheep red cells in vitro, provided mercaptoethanol is present in the culture medium. The mechanism of action of mercaptoethanol under these conditions is not completely clear, but one of its effects is to promote the viability of lymphoid cells in the cultures.

Studies of antibody formation in vitro and in lethally irradiated reconstituted mice. Evidence for an inhibitory function of thymus-derived cells.

The reconstitutive capacity of normal lymphoid cells versus cells obtained from mice immunized with keyhole limpet haemocyanin was studied in lethally irradiated syngeneic recipients using horse erythrocytes as the test antigen. Evidence was obtained for an immune deficiency in the inoculum from immune donors, which was interpreted as evidence in favour of a cellular mechanism to account for antigen‐induced suppression (‘antigenic competition’). The unexpected inhibition of reconstitution with excessive numbers of normal spleen cells was duplicated in experiments using thymus or thymus‐derived cells. Experiments assessing the effect of cortisone‐resistant thymus cells on the response of normal spleen cells, in vivo and in vitro, gave evidence that a relative surplus of such thymus cells possess a capability for suppressing antibody formation. Since helper function by T cells is now well documented, this work is further indicative of the broader view, namely immuno‐regulation by T cells.

Regulation of the immune response by antibody: I. Suppression of antibody formation and concomitant enhancement of cell-mediated immunity by passive antibody

The ability of passively administered antibody to regulate the humoral and cell-mediated immune responses to flagellin, polymerised flagellin, and sheep red blood cell was investigated in rats. It was found that for all three antigens, suppression of antibody formation by passive antibody was accompanied by enhanced delayed-type hypersensitivity. This phenomenon was observed with both homologous and heterologous passive antibody. However, with flagellin and sheep red blood cell, high concentrations of passive antibody led to a decline of delayed-type hypersensitivity. This regulation of immune responses by passive antibody was antigen specific. The delayed-type hypersensitivity induced by antigen in the presence of specific passive antibody persisted for at least 4 weeks after immunisation and was transferable by immune cells but not by serum.

Graft-versus-host reactions in mice. II. Antagonism between marrow and thymus cells

Irradiated (600 R) F1 hybrid mice were grafted with parental thymus and/or marrow cells, and survival time, proliferation, and erythropoiesis in spleen, and content of bone marrow and spleen erythropoietic progenitor cells were determined. Thymus cells alone effected a fatal graft-versus-host reaction (GVHR), and survival times were inversely related to inoculum size. Marrow syngeneic with thymus antagonized this lethal effect. Synergistic splenic proliferation by marrow-thymus cell grafts was observed to be due to enhancement of erythropoiesis and not GVHR. Marrow and spleen progenitor cells were suppressed by grafts of thymus cells alone, but were replenished by marrow-thymus cell mixtures, and were of donor origin. Marrow cells allogeneic with thymus cells failed to protect, to proliferate synergistically, or to replenish progenitor cells. A regimen of cortisol that suppresses antibody formation but not erythropoiesis did not prevent marrow-thymus cell antagonism. Thus, marrow syngeneic with thymus cells antagonizes the GVHR by supplying progenitor cells which are not subject to attack but are stimulated by a small and decreasing host stem cell pool.

Antibody plaque formation in vitro in the presence of respiration inhibitors.

The suppressive effects of 4 respiration inhibitors on <i>in vitro </i>antibody plaque formation to sheep erythrocytes by mouse spleen cells in agar gel was determined. Potassium cyanide, at a concentration of 10<sup>––</sup><sup>2</sup> m, and rotenone, at a concentration of 10<sup>––4</sup> m, markedly suppressed antibody plaque-forming cells (PFC) when added to the agar gel <i>in vitro. </i>In contrast, potassium azide, at a concentration of 10<sup>––2</sup> m showed a slight inhibition and thenoyltrifluoroacetone, at a 10<sup>––3</sup> m-concentration, showed no effect. Both cyanide and azide decreased the hemolytic titers of serum hemolysins at a 10<sup>––2</sup> m-concentration. All inhibitors had no effect on serum hemolysins at 10<sup>––3</sup> m-concentration or lower. These results support the concept that actual viability of antibody-forming cells is not necessary for hemolytic plaque formation <i>in vitro, </i>since only relatively large concentrations of respiration inhibitors, much greater than the dosages needed to inhibit cell metabolism, inhibited antibody formation.

Inhibition of 19S- and 7S-Antibody-Formation by Adoptively Transferred Heterologous and Homologous Immunized Cells

This work was designed to show whether heterologous and homologous sensitized cells are as efficient in suppressing 19S- and 7S-antibody-formation as are isologous sensitized cells. Spleen cells sensitized against sheep red blood cells (SRBC) were collected from guinea pigs, rats and mice of the inbred strains AKR and C57B1. Those cells were transferred to AKR mice one day before sensitization against SRBC. Ten days thereafter spleen cells producing local hemolysis in gel were counted. Controls were AKR mice to which the same number of cells producing antibodies against human red blood cells (HRBC) had been transferred one day before sensitization against SRBC, as well as AKR mice to which the same number of normal cells had been transferred. Another group of AKR mice has not been sensitized after transfer of antibody-producing cells. After transfer of sensitized spleen cells an extreme depression of 19S- and 7S-antibody-formation was found. This effect was demonstrated with cells of the different species. Transfer of spleen cells sensitized against HRBC, as well as of normal spleen cells, caused no significant influence on the primary immune response against SRBC. After transfer of isologous sensitized cells, the background increased. The results show that transfer of heterologous and homologous sensitized cells suppresses antibody formation as efficiently as transfer of sensitized isologous cells.

Biological characterization of an immunosuppressant from group A streptococci.

A component in extracts of Group A streptococci suppresses antibody formation in mice against heterologous erythrocyte and protein antigens. Large doses are not toxic and repeated injection does not change its effectiveness. It is most effective when injected 1 or 2 days before antigen and it is not suppressive when given after antigen. The active factor occurs as a large polydisperse complex and activity can be increased 10- to 25-fold by filtration through Sepharose 2B. Both direct (gammaM) and indirect (gammaG) antibody-forming cells are suppressed in primary and secondary responses. Injection before a primary response does not reduce memory cell development. It increases rather than depresses the "background" antibody-forming cells to sheep erythrocytes, and is equally effective if injected intraperitoneally or intravenously. Ribonuclease increases activity while deoxyribonuclease has no effect. Proteases destroy immunosuppressive action.

Immunotolerance to insulin and Freund's adjuvant.

Prior injections of incomplete Freund's Adjuvant with saline did not suppress antibody formation by ox insulin. This finding is in contrast to that of Jankovic (Colloq. Int. Cent. Nat. Rech. Sci. 116, 187 (1963)), namely that prior injection of complete Freund's Adjuvant suppressed antibody formation to bovine serum albumin. The essential factor in the induction of tolerance to insulin by injection of maleyl insulin in incomplete Freund's Adjuvant is maleyl insulin.

Antibody formation: reduced responses after administration of excessive amounts of nonspecific stimulators.

Enhancement of antibody formation in vitro and in vivo by poly(A.U), cyclic AMP, dibutyryl cyclic AMP, or any one of these agents in combination with theophylline or caffeine, is usually limited to certain dose ranges. High doses of these nonspecific stimulators can produce less or no enhancement, or may actually inhibit antibody formation. Such dose-response relationships may be pertinent to an understanding of phenomena of specific immunological nonresponsiveness and certain types of antigenic competition.

Effects of isopentenyl adenosine and phytohemagglutinin upon RNA syntheses in lymphocytes.

N6-(δ2-isopentenyl) adenosine (iPA) has been previously shown to inhibit DNA and RNA synthesis in cultured lymphocytes and to suppress antibody formation in vivo. The effects have been extended to show that iPA has little preferential action on phytohemagglutinin-transformed spleen cells in vitro. Finally, iPA is found to alter the methylation of all species of RNA but in a disproportionate manner in either PHA-transformed or non-stimulated lymphocytes.

Persistence and spread of influenza virus (A2-Hong Kong) in normal and poly I.C. treated baboons (Papio cynocephalus).

SummaryBaboons inoculated intranasally with influenza virus A2/Phila. 101/68, a Hong Kong strain, excreted virus for as long as 25 days. Infection was associated with seroconversion and transmission to uninoculated control animals. The control animals also excreted virus and showed seroconversion. Treatment of inoculated baboons with poly I.C. delays or suppresses antibody formation and prevents it in uninoculated contact animals. In neither case is virus excretion consistently prevented.

Suppression of antibody formation against Sendai virus in the SV40 and adenovirus 16 infected hamsters.

Золотистых хомячков заражали вирусами SV40 или адено типа 16. Через 9 дней после заражен ия им вводили живой парагриппозны й вирус Сендай. У этих животных иаблюдалось подавле ние антителообразов ания к вирусу Сендай по срав нению с контрольными. В связи с полученными данными кратко обсуж дается проблема иммунодепр ессивного действия о нкогенных вирусов.