Regulation of the primary in vitro response to TNP-polymerized ovalbumin by T suppressor cells induced by ovalbumin feeding

Spleen cells from DBA/2 mice that received a single feeding of 20 mg of ovalbumin (OVA) 7 days previously were specifically hyporesponsive to primary in vitro challenge with the thymic-dependent antigen TNP-polymerized ovalbumin (TNP-POL-OVA). The tolerance observed in spleen cells from OVA-fed animals was dependent upon OVA-specific T suppressor cells, because splenic T cells from OVA-fed mice suppressed the primary response to TNP-POL-OVA of cultures containing normal T and B cells. The tolerance and suppression was OVA specific, because spleen cells from OVA-fed animals responded well to other antigens (including TNP on another carrier), and splenic T cells from OVA-fed mice did not affect the response of normal T and B cells to sheep erythrocytes. These data confirm the existence of T suppressor cells after OVA feeding and provide a direct means of assaying their activity in a primary in vitro response.     

Induction of oral tolerance in the primed immune system: influence of antigen persistence and adjuvant form

Oral tolerance is being promoted as a therapy for autoimmune diseases and therefore will need to be functional in a primed immune system. In previous studies, we found that although primed mice could be tolerized by feeding ovalbumin (OVA), the degree of the tolerance and its effects on individual components of the systemic immune response were more limited than that found in naive animals. Here we increased the dose and frequency of antigen feeding in an attempt to extend the effects of oral tolerance in primed mice and to understand why its effects are limited under these conditions. Increasing the amounts of OVA fed, up to a single dose of 400 mg, or using multiple feeds of 5 x 5 or 5 x 25 mg OVA, did not radically alter the extent of tolerance, with DTH responses, antigen-specific proliferation, and IL5 and IFN-gamma production still being tolerized, but antibody responses remaining generally resistant. The deficient tolerance in primed mice could not be overcome by waiting for maximum clonal expansion to wane and was not influenced by persistent release of antigen from a depot adjuvant. We conclude that the resistance of primed mice to oral tolerance may be due to the fact that antigen-experienced T cells may be inherently resistant to induction of tolerance, or that the microenvironment of the primed immune system inhibits the delivery of tolerogenic signals to antigen-specific T cells.     

Neonatally induced tolerance to soluble protein antigens. I. Analysis of B-cell and T helper cell function in mice tolerant to bovine serum albumin or fowl gamma-globulin

High dose tolerance to either bovine serum albumin (BSA) or fowl γ-globulin (FGG) was induced in CBA mice by neonatal injection. Tolerance to BSA lasted about 9 weeks, and that to FGG, about 18 weeks. Splenic B-cell function was analyzed using quantitative in vivo assays and in vitro limiting dilution analysis. Tolerogen-specific IgM- and non-IgM-producing B cells are depleted at least threefold in the spleens of tolerant mice. Tolerogen-specific T-helper-cell function was examined by immunization with haptenated antigens. Analysis of the recovery from tolerance indicates that the return to normal function in the tolerogen-specific B-cell and T helper fractions coincides with the return to normal responsiveness by the whole animal.     

Production of antibody to homologous -fetoprotein in rabbits, rats and horses by immunization with human -fetoprotein

The production of antibody to homologous alpha fetoprotein (AFP) in rabbits, rats, and horses by immunication with human AFP is reported. The antigens were administered subcutaneously 5 times at intervals of 7-10 days. Rabbits and dogs received 1 mg of human AFP/ml of the homologous pooled newborn serum with each injection while the rats received 1/2 of the dose. The horses received 5 mg/ml/injection. 2 weeks after the last injection, antisera were collected and immunologic assays were performed by the Ouchterlony method and the reversed version of the Mancini method. High titered antibodies were produced in all animals except in the dog. The rabbit, rat, and horse antibodies crossreacted with their own homologous AFP. Attempts to produce antibody with homologous AFP in rabbits, rats, and dogs were unsuccessful.     

Factors affecting production of contrasensitizing antisera in mice.

Using four different protein antigens, two different strains of mice, and various immunization protocols, we have studied production in mice of immunological enhancement antibodies that specifically suppress induction of delayed hypersensitivity. Primary assay of these antibodies was in vivo, because no in vitro test used detected them dependably. Any antigen priming that favored initiation of humoral antibody responses prepared mice to make these contrasensitizing antibodies vigorously following appropriate boosting. The method of boosting usually was more important than that of priming, high titers regularly developing only when primed mice were boosted with much antigen in a short time and were bled a few days later. The presence or absence of delayed hypersensitivity was immaterial. CAF₁ mice made these antibodies better than CF-1 mice, and antigen effectiveness correlated with propensity to induce humoral antibody formation in mice, decreasing from ovalbumin through human serum albumin and bovine serum albumin to methylated human serum albumin. In certain antigenmouse combinations (e.g., ovalbumin in CAF₁ mice) immunosuppressive antibody production was vigorous and prolonged; in others (e.g., bovine serum albumin in CF-1 mice) it was moderate and brief. From our results one can predict what conditions should induce formation of strongly enhancing/contrasensitizing antisera, and speculate that these conditions also should elicit strong, active immunologic tolerance for averting induction of delayed hypersensitivity.     

Regulation of IgE activity in inhalational tolerance via formation of IgG anti-IgE/IgE immune complexes

Background

Allergic asthma is an inflammatory disorder of the airways that results from inappropriate production of IgE against harmless, environmental antigens. Sequestration of free IgE using humanized IgG anti-IgE is an effective therapy for asthma and other atopic disorders. However, the status of free IgE in subjects who have naturally developed immune tolerance to inhaled antigens has not been well studied.

Methods

C57BL/6 mice were sensitized and challenged with ovalbumin (OVA) for 7 days to induce allergic airway disease (AAD) or 6 weeks to induce a state of local inhalational tolerance (LIT). Serum from AAD or LIT mice, diluted to achieve equivalent levels of total OVA-specific IgE, was used to sensitize rat basophil leukemia cells for allergen-mediated degranulation. Levels of degranulation were measured in relation to serum concentrations of free IgE and IgG anti-IgE/IgE immune complexes.

Results

Serum from AAD animals induced a greater degree of basophil degranulation than serum from LIT animals. These results correlated with higher levels of free IgE in AAD animals, whereas LIT mice demonstrated a significant increase in IgG anti-IgE/IgE immune complexes relative to their diseased counterparts.

Conclusions

Sequestration of free IgE by naturally occurring IgG anti-IgE may aid in the development of immune tolerance against inhaled allergens. The decrease in bioavailability of free IgE may, in turn, contribute to the overall reduction of asthma symptoms via a mechanism that mimics the therapeutic effects of humanized IgG anti-IgE.

     

Oral-tolerance induction in diet-induced obese mice

OBJECTIVE: It is known that immune functions are altered in various ways by obesity. However, changes in the intestinal immune system resulting from obesity remain poorly understood. Oral tolerance is a system that suppresses antigen specific immune responses to orally administrated antigens. The intestinal immune system is intimately associated with the oral tolerance system, that acts to prevent allergic and inflammatory diseases. In this study we investigated the effect of obesity on induction of oral tolerance to ovalbumin (OVA) in an animal model of obesity. RESEARCH METHODS AND PROCEDURES: Obese mice induced by a high fat diet and control mice were allowed free access for 3 days to a 1%-ovalbumin (OVA) solution in drinking water. After continuous feeding of the antigen, all the mice were immunized by two intraperitoneal injections of OVA administered 7 days apart. RESULTS: In the control mice, induction of oral tolerance caused an increase in antigen specific IgG1 levels and a decrease in IgG2a levels. In contrast, the IgG1/IgG2a ratio was reversed in obese mice. OVA-specific IL-2 production was suppressed by antigen feeding in both the control and obese mice; however, suppression of OVA-specific IL-10 was observed only in the control mice. Although OVA-specific IgA and IgM were not affected by antigen feeding, the obese groups of mice had significantly lower titers of antibodies. DISCUSSION: These findings suggest that obesity may affect induction of oral tolerance following antigen feeding and that these changes may be related to the inflammatory reaction.     

Immunosuppressive activity of the ethanol extract of Siegesbeckia orientalis on the immune responses to ovalbumin in mice

The in vitro and in vivo immunosuppressive activity of the ethanol extract of Siegesbeckia orientalis (EESO) was studied on the immune responses in mice. EESO significantly suppressed concanavalin A (Con A)- and lipopolysaccharide (LPS)-stimulated splenocyte proliferation in vitro in a concentration-dependent manner. ICR Mice were immunized subcutaneously with ovalbumin (OVA) on days 0 and 14. Beginning on the day of immunization, the mice were administered intraperitoneally with EESO at a single dose of 0.25, 0.5, and 1.0 mg at intervals of 7 days. On day 28, OVA-specific antibodies in serum, and mitogen- and OVA-induced splenocyte proliferation were measured. EESO significantly suppressed Con A-, LPS- and OVA-induced splenocyte proliferation in the OVA-immunized mice in a dose-dependent manner. The OVA-specific serum IgG, IgG1, and IgG2b levels in the OVA-immunized mice were also significantly reduced by EESO. Moreover, reducing effect on the IgG1 antibody of EESO at the dose of 1.0 mg was more significant than that of cyclosporin A (CsA; positive drug). The results suggest that EESO could suppress the cellular and humoral response to ovalbumin in mice, and deserve further investigations to be developed as immunosuppressant.     

Immune response in dog. 3. Antibody formation in dogs and rabbits to human serum proteins and keyhole limpet haemocyanin.

Antibody formation in dogs and rabbits to human serum proteins and keyhole limpet haemocyanin (KLH) following primary, secondary and multiple stimulation was analysed. Primary injection of human serum stimulates the formation of precipitin antibodies to beta2 lipoprotein in dogs, beta2 lipoprotein and beta globulin in rabbits. After a secondary dose the rabbits formed precipitins to a whole range of human serum proteins, while the dogs to beta2 lipoprotein, albumin, beta globulin. When the primary dose of antigen was divided over a period of 8 weeks, the dog produced precipitins to beta2 lipoprotein while the rabbit to a wide range of serum proteins. Secondary stimulation of these animals did not increase the number of precipitins formed. Quantitative analysis of the antibody produced show that the best response was with beta2 lipoprotein followed by albumin and beta globulin. As the immunogenicity of the antigen was greater the differences between the two species were narrow. These differences were less pronounced following the primary injection than after the secondary and multiple stimulations. The primary response to KLH (which represents mainly IgM) is better in the dog than in the rabbit, while the secondary response (IgG) was better in the rabbit. The poorer IgG response in the dog compared to the rabbit, observed in all the experiments, is discussed.     

The role of T cells in IgG production; thymus-dependent antigens induce B cell memory in the absence of T cells.

B cell memory was shown to develop in congenitally athymic (nu/nu) mice after injection with small amounts of thymus-dependent antigens, in particular heterologous serum proteins, such as fown gamma-globulin (FGG) or DNP-bovine-serum albumin (DNP-BSA). Large doses of proteins (10 mg) tended to produce a specific B cell unresponsiveness, although there was still some evidence of B cell priming. The antigen did not have to be in a multivalent form to interact with B cell so as to induce immunologic memory or tolerance. In contrast to the induction of B cell memory, the production of IgG antibody in this system was found to be strongly T cell dependent. Thymus-independent antigens like LPS or POL with pronounced adjuvant effects on IgG production in normal or surgically thymectomized mice, could not replace T cells in allowing an IgG response against thymus-dependent antigens in congenitally athymic mice. However, the action of T cells once activated is likely to be non-antigen-specific, since it was shown that supernatants of antigen-activated-syngeneic T cells stimulated IgG production in cultures of primed B cell populations non-antigen-specifically.     

Control of IgE and IgG1 antibody production in mice.

The production of IgE and IgG1 was studied in untreated, thymectomized. splenectomized, anti-thymocyte serum-treated, or sublethally X-irradiated mice. Dinitrophenyl Ascaris and ovalbumin were used as antigens, and aluminum hydroxide was used as adjuvant. A suppression of IgE production was observed in adult thymectomized mice, although the kinetic pattern of the antibody response was the same as in control animals. IgG1 antibody production was not affected by thymectomy. Splenectomy did not change either IgE or IgG1 production. A single dose of rabbit anti-thymocyte serum (ATS) given 8 days after immunization inhibited IgE antibody production. The effect of ATS was dose dependent and also varied with the amount of antigen used, the immune response to high doses being more susceptible to the effect of ATS. No alteration in IgG1 production was caused by ATS even when IgE antibody formation was completely inhibited. When preceding immunization, sublethal irradiation enhanced IgE antibody formation and partially suppressed IgG1 production; applied after immunization, irradiation caused an enhancement of IgE production which was inversely proportional to the interval elapsed between the two procedures. On the other hand, the IgG1 antibody production was fairly resistant to the same treatment. The results suggest a clearcut separation between the mechanisms regulating IgE and IgG1 production in mice.     

Hypophysectomy and neurointermediate pituitary lobectomy decrease humoral immune responses to T-independent and T-dependent antigens

In rats, hypophysectomy (HYPOX) or neurointermediate pituitary lobectomy (NIL) reduce humoral and cell-mediated immune responses. However, to our knowledge, the differences in the effects of anterior versus posterior pituitary hormones on the immune responses have not been studied to date. We compared in rats, the effects of sham surgery (SHAM), HYPOX, and NIL on humoral immune responses to T cell-independent (TI) type 1 antigen DNP-LPS and to TI type 2 antigen DNP-FICOLL, as well as to T cell-dependent (TD) antigens ovalbumin (OVA) and bovine serum albumin (BSA). The results showed that: (1) both HYPOX and NIL induced a similar and significant decrease in IgM responses towards TI-1 antigens, (2) NIL but not HYPOX induced a decreased IgM response to TI-2 antigens, and (3) both HYPOX and NIL induced similar and significant decrease in IgG responses to TI-2 antigens. Compared with the SHAM group, IgM responses to both TD antigens did not change in HYPOX and NIL animals, whereas the IgG responses to OVA and BSA significantly decreased in HYPOX and NIL animals. These results indicate that hormones of the anterior and posterior pituitary play their own role in the regulation of humoral immune responses.     

Nucleoside specificity in the carrier IgG-dependent induction of tolerance.

Induction of tolerance to nucleoside haptens in BALB/c mice with isologous IgG conjugates bearing four nucleosides simultaneously (A, G, C, T)-IgG was confirmed. A mixture of separate nucleoside-IgG tolerogens (A-IgG, G-IgG, C-IgG, and T-IgG) was as effective or more effective that the (A, G, C,T)-IgG form in suppressing the response to (A, G, C, T)-KLH. The nucleosides acted independently and simultaneously, since tolerogens with varying combinations of nucleosides caused specific suppression of the respones to only those nucleosides present on the tolerogen. Nucleoside-IgG conjugates did not suppress the response to denatured DNA-methylated bovine serum albumin, in which larger oligonucleotide determinants predominate. In varying combinations, guanosine was the dominant nucleoside both for immunization and for induction of tolerance. After three or four immunizations, control immunized animals made mainly IgG anti-nucleoside antibodies and this IgG antibody formation was preferentially suppressed in tolerogen-treated animals. Tolerance could be established before the primary or secondary immunization and it then persisted for at least 75 days through a fourth course of immunization. The same dosage of tolerogen did not reverse a strongly established anti-nucleoside antibody production after a tertiary response.     

An investigation of tolerance to the actions of leptogenic and anorexigenic drugs in mice

This study compared the effects of chronic administration of anorexigenic drugs on weight loss in mice. Tolerance to the effects of peripheral anorexigenic peptides, viz. cholecystokinin-octapeptide and bombesin, developed rapidly. Morphine, cocaine and dehydroepiandrosterone-sulfate caused weight loss and appeared similar to d-amphetamine in mechanisms of action. A high dose of fluoxetine (25 mg/kg) proved to be a potent leptogenic agent but was also associated with death in some animals. A lower dose of fluoxetine (5 mg/kg) was associated with the development of tolerance. Calcitonin, a potent anorexigenic agent, did not produce weight loss and tolerance to its anorectic effect had developed by 10 days. Animals varied widely in their individual responsiveness to a given drug. Peripheral administration of peptide YY caused weight loss. We conclude that acute or chronic effects of agents on food intake do not necessarily predict effects on body weight. However, neurotransmitters that enhance feeding centrally appear to cause weight loss when administered peripherally.     

Protracted circulating lifetimes of mannose-terminated glycoproteins and aggregated albumin in mice infected with LDH-elevating virus.

Several macromolecular homeostasis-regulating mechanisms were tested for functional integrities in mice during acute and early chronic phases of infection with lactic dehydrogenase-elevating virus (LDV). Fractional catabolic rates of carbodiimide-aggregated albumin and immunoglobulin G were studied to evaluate glomerular filtration and hepatic Kupffer cell phagocytic activities. Several glycoproteins (fetuin, IgG antibodies, and ovalbumin) were also compared with their deglycosylated counterparts for fractional catabolic rates and organ distributions as a basis for evaluating virus-induced modifications of saccharide-binding "receptor functions" in vivo. Findings were that normal hepatic clearance of aggregated albumin and of ovalbumin is slowed from the onset of viremia. Fractional catabolic rates of amannosyl-ovalbumin and amannosyl-IgG are similar in uninfected animals to those seen with native ovalbumin or with mannose-terminated IgG in LDV-infected animals. Ovalbumin and aggregated albumin were also found to be mutually competitive for hepatic uptake in uninfected animals. It is proposed that LDV, which replicates in cells of the mononuclear phagocyte system (reticuloendothelial system), alters the clearance functional state of fixed tissue macrophage, thereby explaining in part the protracted circulatory longevity of several enzymes, aggregated albumin and mannose-terminated ovalbumin, and IgG in LDV-infected mice.     

Immunosuppressive activity of Semen Persicae ethanol extract on specific antibody and cellular response to ovalbumin in mice

The immunosuppressive activity of the ethanol extract of Semen Persicae (EESP) was studied with respect to specific antibody and cellular response to ovalbumin (OVA) in mice. The effects of EESP on mice splenocyte proliferation in vitro were measured. EESP significantly suppressed concanavalin A (ConA)- and lipopolysaccharide (LPS)-stimulated splenocyte proliferation in vitro in a concentration-dependent manner. Furthermore, the effects of EESP at three dose levels on the humoral and cellular immune responses in the OVA-immunized mice were examined. ICR Mice were immunized subcutaneously with OVA on day 0 and 14. Starting on the day of immunization, the mice were administered intraperitoneally with EESP at a single dose of 0.25, 0.5, and 1.0 mg, and cyclosporin A (CsA, positive drug) at a single dose of 0.1 mg at intervals of 7 days. On day 28, mitogen- and OVA-induced splenocyte proliferation and OVA-specific antibody level in serum were measured. EESP significantly decreased ConA-, LPS-, and OVA-induced splenocyte proliferation in the OVA-immunized mice at the dose of 1.0 mg. Meanwhile, the OVA-specific serum IgG, IgG1, and IgG2b antibody levels in the OVA-immunized mice were markedly reduced by EESP in a dose-dependent manner. The results suggest that EESP could suppress the cellular and humoral immune response in mice, and deserve further research to be developed as immunosuppressant.     

Separation of antigens by immunological specificity. Studies on the desorption of homologous antigen from disulphide-linked antibody immunosorbents

1. Glycine-hydrochloric acid buffer, pH2.2, desorbed (131)I-labelled human serum albumin (100%), lysozyme (100%), ovalbumin (90%), fluorescent ovalbumin (50-60%) and fluorescent human gamma-globulin (20%) from their respective homologous disulphide-linked antibody immunosorbents; reasons are suggested for the low recoveries of the fluorescently labelled proteins. 2. Approx. 40% of the recovered (131)I-labelled human serum albumin and fluorescent ovalbumin was desorbed above pH6.0, but lysozyme was not eluted until the pH was 3.0 or below. 3. In all cases where high recoveries of antigen were obtained, the immunosorbents could be regenerated and recycled at least four times with full retention of specificity and minimal diminution of capacity. 4. The desorbed antigens were unchanged when compared with the original antigens by quantitative precipitin, specificradioactivity, fluorescent and enzymic analyses and by cellulose acetate electrophoresis. 5. Desorption of antigen with a variety of reagents was investigated. These reagents were less satisfactory than glycine-hydrochloric acid.     

Elimination of intestinally absorbed antigen into the bile by IgA

Polymeric serum IgA has been shown to transport antigens in the form of IgA immune complexes from the circulation into the bile. To test the hypothesis that this mechanism could aid in the clearance of intestinally absorbed antigens, mice were gastrically intubated with trinitrophenylated human serum albumin (TNP-HSA). After 3 hr, two groups were injected i.v. with either MOPC 315 (IgA, anti-TNP) or control ascitic fluid. One hour later, blood and bile samples were taken. Small amounts of antigen (0.0008% of the intubated dose) were detected in the serum of control animals by solid phase radioimmunoassay. In animals receiving anti-TNP IgA, serum levels were decreased significantly and intact antigen appeared in the bile, whereas no antigen was detectable in the bile of control mice. These results suggest that IgA-mediated hepatobiliary transport can function as one of the mechanisms for the elimination of intestinally absorbed antigens.     

Separation of antigens by immunological specificity: Use of cellulose-linked antibodies as immunosorbents

1. Immunosorbents were prepared by coupling activated aminocellulose with the γ-globulin concentrates of antisera prepared against ovalbumin and human serum albumin. 2. The immunosorbents were low in solubility, but high in capacity for homologous antigens. 3. The high specificity of these immunosorbents was demonstrated by their use in fractionating various mixtures of fluorescent ovalbumin, ¹³¹I-labelled human serum albumin, lysozyme and ribonuclease.     

Potentiation of the rat delayed-type hypersensitivity reaction by the Fc portion of human IgG1

Fc fragments derived from a human IgG1 myeloma protein potentiate the rat delayed-type hypersensitivity (DTH) reaction to antigen challenge. Lewis rats immunized with heat-killed tubercle bacilli give augmented DTH reactions to the purified protein derivative of tuberculin when Fc fragments are included in the challenge dose. Similar potentiation of DTH by pFc' fragments indicates that the active site is located in the CH3 domain of IgG1. Histologic evaluation of the augmented reaction sites revealed predominantly mononuclear cell infiltrates characteristic of DTH reactions. Skin tests of tubercle bacilli-sensitized rats with an unrelated antigen and/or Fc fragments fail to elicit significant reactions. Augmentation of the DTH reaction to purified protein derivative is restricted to the Fc or pFc' region fragments since intact monomeric IgG1, Fab fragments, and bovine serum albumin were all shown not to be active potentiators. The DTH reaction of ovalbumin-sensitized rats was similarly augmented when Fc fragments were included with a challenge dose of ovalbumin, thus supporting the general nature of the phenomenon. These results support the concept of Ig molecules as multifunctional proteins that can not only serve effector functions but also participate in the regulation of immune responses.