Anaphylactic hypersensitivity in isolated smooth muscle preparations from adjuvant-treated guinea-pigs

Anaphylactic hypersensitivity of isolated guinea-pig ileum preparations has been evaluated by investigation of antigen-dose/response relationships, in vitro. The influence of Freund's adjuvants on active sensitization has been investigated. In contrast to the inhibitory influence previously observed in vivo, the degree of hypersensitivity of isolated tissues in vitro was potentiated by the adjuvants. It is thought that high circulating titres of antibody in vivo exerted a protective or neutralizing effect in relation to challenging doses of antigen. Isolated tissues were freed from this influence. Tissues sensitized with the aid of Freund's complete adjuvant (FCA) revealed the influence of other inhibitory mechanisms, due probably to interaction of various antibody types.     

Lesions in Escherichia coli cell walls caused by the action of mouse complement

Characteristic lesions formed by mouse complement were visible in electron micrographs of Escherichia coli cell walls treated with aminoethylisothiouronium bromide and then incubated with normal mouse serum in vitro. However, lesions could not be detected in E. coli cell walls recovered from the blood of normal mice after intravenous injection.

Lesions were not produced by either heated or complement deficient mouse sera in any of the experiments.

After incubation with normal or complement deficient mouse serum E. coli cell walls were surrounded by a layer probably made up of complement components and immunoglobulins. This layer was less evident when the sera had been heated at 56° for 1 hour.

Numerous lesions formed by the in vivo action of guinea-pig complement were found in E. coli cell walls recovered from the animal after intravenous injection.

     

Stimulation of humoral and cell mediated immunity by polysaccharide from mushroom phellinus linteus

Polysaccharide was purified from mycelial culture of Phellinus linteus (PL) and its effect on immunocompetence of normal splenocytes was observed. Overall in vitro and in vivo immune function was enhanced by addition of PL into culture of mouse spleen lymphocyte and I.p. injection into mouse, while β-glucans and other polysaccharides only raised the level of T lymphocyte-mediated immunity. PL stimulated immune functions of T lymphocytes, such as proliferation of T lymphocyte induced by mixed lymphocyte reaction and cytotoxicity of cytotoxic T cells responding to alloantigen. Nonspecific immune functions mediated by natural killer cells and macrophages were increased by treatment of PL in vivo and in vitro. PL also stimulated humoral immune function positively, such as T-dependent and T-independent primary antibody responses, and acted as a polyclonal activator on B cell. PL exhibited a wider range of immunostimulation and antitumor activity than other polysaccharides isolated from Basidiomycetes.     

β Toxin of Clostridium welchii : Cytotoxic Effects Produced by the • on Guinea-Pig Monocytes

The cytopathic effects of Cl. welchii β toxin on guinea-pig monocytes in vitro have been studied using the uptake of eosin-Y^* as evidence of cell death.

The experiments show that these effects are due to antigen—antibody reaction probably on the cell surface, and that these reactions are not dependent on the presence of complement. Monocytes can be actively sensitized in vivo, and passively sensitized in vitro. The serum used to passively sensitize the monocytes need not possess a precipitating antitoxin titre.

Comparable experiments using an ovalbumin antigen—antibody system produced the same cytopathic effects on the monocytes as those which occurred in the β toxin—cellular system.

     

Antigenic correlations between brain and thymus. I. Common antigenic structures in rat and mouse brain tissue and thymocytes

Rabbit anti-mouse brain serum (AMBS) and anti-rat brain serum (ARBS) were found to exhibit cytotoxic activity against homologous thymocytes. AMBS also proved to be cytotoxic for rat thymocytes and ARBS for mice thymocytes. Although rat lymph node and spleen cells were nearly uneffected by ARBS, AMBS exhibited some cytotoxicity against the lymph node and spleen cells of mice. The percentage of mouse lymph node and spleen cells lysed by AMBS resembled that caused by Θ-isoantiserum, Nevertheless, unlike Θ-isoantisera, the AMBS was also cytotoxic for AKR thymocytes. Absorption analyses showed that the cytotoxic activity on mouse thymocytes could be absorbed out of the ARBS by both rat and mouse thymocytes and brain residues, However, the latter failed to remove cytotoxic effects on rat thymocytes. Conversely, rat thymocytes and brain homogenate did not influence the cytotoxic effects of AMBS on mouse thymocytes, but eliminated its cytotoxic effects on rat thymocytes. After previous in vitro treatment with anti-CBA mouse brain serum or ARBS, CBA brain residue proved no longer capable of absorbing any Θ-C3H isoantibodies. This indicates that the antibrain antibodies had blocked the Θ-determinants.     

Macrophage cytotoxicity factor. A product of in vitro sensitized thymus-dependent cells

Peritoneal mouse macrophages are rendered specifically cytotoxic to allogeneic target cells by a cell-free filtered supernatant from in vivo sensitized spleen cells. Pretreatment of sensitized spleen cells with anti-θ serum and complement completely abolishes secretion of the supernatant factor. The factor can also be induced by in vitro sensitization of mouse spleen cells to alloantigens. Furthermore, a cortisone-resistant population of pure thymocytes can be sensitized in vitro and induced to liberate the factor. The macrophage cytotoxicity factor is only effective if bound to macrophages and can be transferred to iso- and allogeneic macrophages without loss of activity.     

Antilymphocyte antibody purified by immunoabsorption and elution

A comparison has been made of several techniques for the preparation from antilymphocyte serum of pure antilymphocyte antibody by absorption on, and elution from mouse thymocytes. The best yield was obtained by a batch method using cells fixed in 0·1% formaldehyde as immunoabsorbent followed by elution with 0·12 M citrate buffer at pH 3·0. The antilymphocyte activity of eluates was studied in vitro by determination of membrane immunofluorescence titres and in vivo by the neutralization of the local renal xenogeneic graft-versus-host reaction. These tests showed that an IgG eluate of a rabbit antilymphocyte serum was nineteen times more active than the same weight of the whole IgG present in the parent serum.     

Role of macrophages in tumour immunity: III. Co-operation between macrophages and lymphoid factors in an in vitro allograft situation*

Normal mouse peritoneal macrophages were rendered cytotoxic towards target cells in vitro by incubation with immune C57B1 spleen cells taken early (7 days) and late (21 days) after a single injection of allogeneic DBA/2 lymphoma cells (L5178Y). This process, termed arming, was shown to be immunologically specific. X-irradiation of late immune spleen cells reduced their arming potential, but not that of early cells. Late immune spleen cells only were found to release spontaneously a factor which rendered macrophages weakly cytotoxic in vitro, and this release was not affected by irradiation. Both early and late immune spleen cells could be stimulated to release an arming factor by incubating them with irradiated lymphoma cells and this factor rendered macrophages strongly cytotoxic. Allo-immune serum also armed normal macrophages. Peritoneal macrophages harvested at 7 and 21 days from immunized mice were strongly cytotoxic towards the target cells. The results suggest pathways by which macrophages might become specifically cytotoxic in vivo and also integrated into the general immune response seen during rejection of the allogeneic L5178Y lymphoma.     

Thiol compounds inhibit mercury-induced immunological and immunopathological alterations in susceptible mice

In vitro mercury induces a high proliferative response in splenic lymphocytes and in vivo it induces a systemic autoimmune disease in susceptible mouse strains. This disease is characterized by increased serum levels of IgE and IgG1 antibodies, by the production of anti-nucleolar antibodies and by the formation of renal immune complex deposits. We have previously found that the presence of 2-mercaptoethanol (2-ME) inhibited mercury-induced cell proliferation in vitro. In this study, we tested the effects of four other thiol compounds, namely dithiothreitol (DTT), l-cysteine, meso-2,3-dimercaptosuccinic acid (meso-DMSA) and 2,3-dimercapto-1-propanesulfonic acid, Na salt (DMPS) on mercury-induced immunological changes both in vitro and in vivo. We found that in vitro, the addition of all thiol compounds abrogated mercury-induced cell aggregation and proliferation. In vivo, injection of meso-DMSA and/or DMPS (s.c. or i.p.) immediately following exposure to mercury markedly decreased IgG1 synthesis in spleen cells and serum IgE levels in mercury-susceptible SJL mice. Treatment with DMPS also prevented mercury-induced IgG1 anti-nucleolar antibody synthesis and the development of mesangial IgG1 immune complex deposits in SJL mice.     

The function of T Cells Carrying Receptors for Complexes of Ig and Antigen

Thymocytes exposed briefly in vitro to a variety of particulate substances (such as mycobacteria, erythrocytes of allogeneic cells) or to substances known to act in vivo as adjuvants (LPS or poly A:U), generate supernates which are able to induce cytophilic Ig in normal mouse serum in the presence of a foreign protein (antigen). This cytophilic Ig is taken up by 20–25% of splenic T cells. Hydrocortisone resistant thymocytes show the same property, while bone marrow cells are inactive. This activity is similar to that reported previously as being present in the 4S fraction of mouse serum, collected 6 hours after injection of complete Freund's adjuvant. It is proposed that this factor is responsible for the formation of complexes of Ig and antigen which have been detected in the serum 6 hours after immunization. Thymocytes collected 6 hours after priming in vivo with SRBC (when a subpopulation among them carries easily demonstrable surface Ig) are able to amplify markedly the antibody response particularly the 7S. It is postulated that the factor by generating the cytophilic Ig (complexes ?) which is taken up by T cells, sets up a mechanism which markedly amplifies their helper cell function.     

Effects of antimacrophage, antithymocyte, antilymphocyte and antispleen serum on the immune response in mice

The effect of antimacrophage serum (AMS) on antibody production was compared to that of antithymocyte (ATS) and antilymphocyte (ALS) serum. All three types of antisera inhibited antibody production to SRBC in mice. Antispleen serum was not immunosuppressive. Immunosuppression could best be demonstrated when the antisera were injected 3 days before a low dose of antigen (10⁷ or 5 × 10⁷ SRBC). None of the antisera affected secondary antibody production.

There was no correlation between the immunosuppressive potency of the antisera and their in vitro cytotoxic or in vivo lymphopenic acitivity. AMS inhibited phagocytosis, whereas ATS and ALS enhanced phagocytosis. So far we have been unable to absorb out immunosuppressive activity of the antisera but have been able to absorb out cytotoxic activity. The significance of these findings is discussed.

     

The demonstration of the involvement of antibody in the in vitro lysis of chicken erythrocytes by allergized spleen cells

The experiments described demonstrate that the in vitro lysis of chicken erythrocytes (CRC) by allergized mouse spleen cells (ASC) is an antibody-dependent phenomenon. Supernatants obtained from cultures of ASC were shown to contain antibody which could effect antibody-dependent cell-mediated cytotoxicity.

The cytolysis observed with ASC was inhibited by both an IgG fraction and a F(ab')₂ portion of rabbit anti-mouse IgG. No inhibition was obtained by pretreating the ASC with AKR anti-C3Hθ serum and guinea-pig complement.

     

The adherence to Salmonella enteritidis of IgG, IgM and β1C-globulins from guinea-pig serum

The adherence of globulins from guinea-pig serum to Salmonella enteritidis was determined when these bacteria were sensitized in vitro: (a) in serum from normal guinea-pigs lacking detectable bactericidal antibody, (b) in serum from normal guinea-pigs possessing low titre of bactericidal antibody, and (c) in serum from guinea-pigs actively immunized against this Salmonella. Sensitized bacteria were washed free of contaminating serum constituents and used to immunize rabbits. Guinea-pig globulins that adhered to the bacteria and to which the rabbit responded by the production of antibody were identified by immunoelectrophoresis. IgM, but no IgG, attached to bacteria sensitized in serum lacking bactericidins. Both IgG and IgM attached to bacteria sensitized in immune serum or in serum containing `normal' bactericidins. β_1C-globulin attached to bacteria in all three cases.     

Studies of allograft immunity in mice. II. Mechanism of target cell inactivation in vitro by sensitized lymphocytes

The mechanisms of the in vitro interaction of sensitized lymphocytes and allogeneic target cells has been studied in a tumour allograft system in inbred mice. The cytotoxic effect of sensitized lymphocytes is shown to require the presence of Ca^{+ +} and Mg^{+ +}. Pretreatment of the lymphocytes with trypsin led to inhibition of cytotoxicity, followed by spontaneous reversal after 1–3 hours incubation. Reactivation was found to be blocked by an inhibitor of protein synthesis (cycloheximide). Cortisone was not found to inhibit the lytic interaction significantly; an occasional effect is thought to be due to toxicity of cortisone for lymphocytes as revealed by dye exclusion test. Inhibition of DNA-synthesis with FUdR (an inhibitor of the enzyme thymidine synthetase) did not reduce the lytic activity of sensitized lymphocytes.

Isologous anti-target cell sera induced in various strains of inbred mice were found to be ineffective in blocking the cellular immune reaction in vitro when directed against a minor part of the antigenic complex, but strongly inhibitory when reactive against a major part or the whole complex. Similarly, target cells lacking several of the sensitizing H-2 antigens were not lysed. An isologous anti-lymphocytic serum induced in the graft donor strain and directed against the recipient strain (lymphocyte donor) did not inhibit the cytotoxic reaction. In a heterologous system on the other hand, the lytic effect of guinea-pig lymphocytes sensitized against mouse target cells was effectively blocked by an anti-lymphocytic serum induced in mice of the graft donor strain by injection of recipient (guinea-pig) spleen cells.

     

Antibodies targeting dengue virus envelope domain III are not required for serotype-specific protection or prevention of enhancement in vivo

The envelope (E) protein of dengue virus (DENV) is composed of three domains (EDI, EDII, EDIII) and is the main target of neutralizing antibodies. Many monoclonal antibodies that bind EDIII strongly neutralize DENV. However in vitro studies indicate that anti-EDIII antibodies contribute little to the neutralizing potency of human DENV-immune serum. In this study, we assess the role of anti-EDIII antibodies in mouse and human DENV-immune serum in neutralizing or enhancing DENV infection in mice. We demonstrate that EDIII-depleted human DENV-immune serum was protective against homologous DENV infection in vivo. Although EDIII-depleted DENV-immune mouse serum demonstrated decreased neutralization potency in vitro, reduced protection in some organs, and enhanced disease in vivo, administration of increased volumes of EDIII-depleted serum abrogated these effects. These data indicate that anti-EDIII antibodies contribute to protection and minimize enhancement when present, but can be replaced by neutralizing antibodies targeting other epitopes on the dengue virion.     

Effect of allogeneic cell interaction on the primary immune response in vitro. Cell types involved in suppression and stimulation of antibody synthesis

A graft-versus-host (GVH) reaction was induced in F1 hybrid mice by the inoculation of spleen cells from one of the parental strains. One week later the spleen cells from the recipients were cultured during the conditions for obtaining a primary immune response in vitro described by Mishell & Dutton (1967). It was found that the antibody response against the thymus-dependent antigen sheep red cells (SRC), as well as the thymus-independent antigen lipopolysaccharide from Escherichia coli 055:B5 (CPS) was markedly depressed. Spleen cells from mice subjected to a GVH reaction (GVH cells) also inhibited the antibody response of normal cells in vitro. The inhibitory effect of the GVH cells on normal cells was not sensitive to treatment with anti-θ serum, but could be completely abolished by treatment with iron powder, which removes adherent cells.

By culturing cells of two different mouse strains together in vitro it was possible to obtain stimulation or inhibition of the antibody response depending on the total cell number per dish.

The relation of these results to the phenomenon of antigenic competition is discussed. It is suggested that antigenic competition is caused by a non-specific inhibition of cell proliferation, possibly mediated by a locally acting factor. Both thymus-derived and bone marrow-derived cell lymphocytes are affected by the phenomenon. The cells initially responsible for the inhibition seem to be antigen-activated thymus-derived cells (T-cells), which by secondarily activated cells such as macrophages inhibit other cells. A GVH reaction, which generally leads to antigenic competition may, when less pronounced, cause stimulation of the antibody response.

     

The bacteriostatic effect of serum on Pasteurella septica and is abolition by iron compounds

Methods are described for controlling the oxygen tension and pH of bacterial cultures in vitro. In fresh normal rabbit serum or plasma at a Po₂ of 80–90 mmHg and a pH of 7.5, the presence of specific antibody has a powerful bacteriostatic effect against Pasteurella septica. It is suggested that the serum components involved in this reaction consist of transferrin, specific antibody, and complement. Bacteriostasis can be abolished by a variety of iron compounds. This evidence together with that obtained in vivo (Bullen, Wilson, Cushnie and Rogers, 1968a) strongly suggests that an interference with bacterial iron metabolism plays an important role in resistance to infection against P. septica.     

Cytophilic antibody in guinea-pigs with delayed-type hypersensitivity

Cytophilic antibody has been demonstrated in the serum of guinea-pigs injected with sheep red cells mixed with Freund's complete adjuvant. Sheep red cells adhered, in the absence of serum, to normal guinea-pig macrophages which had been treated in vitro with such serum. Treatment of polymorphs and lymphocytes with the antiserum did not confer on them this capacity to adsorb sheep red cells. The animals immunized with sheep red cells and complete adjuvant, when skin tested with an extract of sheep red cells, showed strong delayed-type reactions.

Cytophilic antibody was not detectable in the serum of the majority of guinea-pigs which received sheep red cells mixed with incomplete adjuvant. Skin tests in these animals produced strong Arthus reactions but no delayed reactions. Titres of haemagglutinating antibodies were similar whether the adjuvant was complete or incomplete.

     

Apoptosis induced by γ-irradiation,but not CD4 ligation,of peripheral T lymphocytes in vivo is p53-dependent

Mice generated by homologous recombination which carry a large deletion of the p53 tumour suppressor gene have a high incidence of spontaneous Thy1-positive thymic lymphoma. Extra-thymic lymphomas are rare. Apoptosis following γ-irradiation in thymocytes from these animals in vitro is p53-dependent and there is a marked gene dose effect: heterozygotes show partial resistance to irradiation-induced cell death. Apoptosis in the T-cell zones of lymph nodes following in vivo γ-irradiation was p53-dependent, but the gene dosage effect was less marked than that noted for thymocytes. Apoptosis was induced in vivo by ligation of CD4 on the cell surface following intravenous injection of anti-CD4 monoclonal antibody. Apoptosis was counted in lymph node sections using a semi-automated morphometric system. This showed no evidence of p53 dependency. In contrast to a previous report, which used a different line of p53-deficient mice, splenocytes from p53-null mice did not differ significantly from wild-type cells with respect to in vitro proliferative activity and response to mitogenic stimulation by concanavalin A. This may be due to strain differences. Therefore, whilst p53 has a role in the deletion of lymphocytes which have acquired pathological DNA strand breaks which may lead to mutations, the results of this study imply that p53 is not involved in the control of apoptosis following engagement of surface receptors, nor in response to physiological DNA breaks and normal recombination events during T-cell ontogeny. © 1997 John Wiley & Sons, Ltd.     

Induction and suppression of the cytotoxic activity of human lymphocytes in vitro by heterologous anti-lymphocyte serum

Heterologous anti-lymphocyte sera were demonstrated to induce a cytotoxic potential in normal non-immunized human lymphocytes against allogeneic fibroblast target cells. The cytotoxicity-inducing capacity was restricted to certain dilutions of anti-lymphocytic serum above and below which no cytotoxic effect was obtained. This optimal concentration shifted towards higher dilutions in sera taken late during the immunization course. The antisera were shown to stimulate the DNA-synthesis in lymphocytes and to aggregate the lymphocytes to the target cells. The DNA-synthesis and the aggregation as well were maximal at the same dilution of anti-lymphocytic serum which induced cytotoxicity. No cytotoxic effect was demonstrable on sheep fibroblasts. It is, therefore, suggested that the anti-lymphocytic serum antibody induces lymphocyte-mediated cytotoxicity against allogeneic fibroblasts in a two step manner: it stimulates the lymphocytes into a cytotoxic state; it aggregates the human lymphocytes to the human fibroblasts by virtue of its bivalent structure.

Anti-lymphocytic serum was also found to suppress the cytotoxic activity of lymphocytes induced by various non-specific agents, such as phytohaemagglutinin, streptolysin O and anti-lymphocytic serum itself. The mechanism for this inhibition is extensively discussed and it is suggested that anti-lymphocytic serum suppresses the reaction by coating the lymphocytes, thereby preventing the intimate contact between effector and target cell. A similar mechanism may operate in vivo and could be a partial explanation of the in vivo immunosuppressive effect of anti-lymphocytic serum. Purified 7S γ-globulin possessed all activities of the whole antiserum.