Lifelong Maintenance of Oral Tolerance and Immunity Profiles in Mice Depends on Early Exposure to Antigen.

Oral tolerance is defined as a state of systemic hyporesponsiveness to an antigen that has been previously administered by the oral route. Many factors affect oral tolerance induction; some of them related to antigen, and some related to the animal. The age of the animal is one of the most important factors that affect oral tolerance as ageing brings many alterations in immune responses. Herein, we demonstrated that both the oral tolerance and pattern of immune reactivity triggered in early life were kept up to 15 months regarding the magnitude of antibody production, cell proliferation and cytokine profile when compared to immune responses induced in old mice. Therefore, our results corroborate with a promising proposal for prevaccination during childhood and young age, and a booster in older age, to make sure that the primary immunization in early life is not lost in aged individuals.

H-2-linked genetic control of immune responsiveness to ovalbumin and ovomucoid.

Immune responsiveness of inbred mice to low doses of ovalbumin or ovomucoid is under control of single dominant genes closely linked to alleles of the H-2 locus. High responsiveness to ovomucoid is linked with the H-2(a) and H-2(k) alleles, and to ovalbumin with the H-2(b), H-2(d), and H-2(q) alleles.

Specific immune responses but not basal functions of B and T cells are impaired in aged mice.

Senescence is characterized by several alterations in the immune system. Such modifications can be found in lymphoid organs as well as in the cellular components of the immune system. Several reports have suggested that immune dysfunction can affect both T and B cells, but T cells have been shown to be more susceptible to the effects of aging. B cell function may also be altered with reduction in germinal center formation, antibody response, and affinity maturation of antibodies. Herein we showed that although antigen-specific antibody response to a soluble antigen declines in 18-month old mice, total levels of serum antibodies as well as frequencies of spleen and bone marrow antibody-producing cells are increased in aged mice. In addition, proliferative response of non-stimulated spleen T cells from aged mice were augmented and insensitive to increasing doses of concanavalin A stimulation as compared to young mice that showed a typical dose-dependent response to mitogen stimulation in vitro. These data suggest that the higher activation mode of B and T cells in senescent mice is a result of an increased frequency of cells committed to previous antigenic experiences and with poor ability to respond to novel antigenic challenges.

Immune responses of inbred mice to repeated low doses of antigen: relationship to histocompatibility (H-2) type.

Immunization of inbred strains of mice with repeated minute doses (0.1 to 1.0 microgram) of hapten-protein conjugates demonstrated wide differences in the magnitude of their antibody responses, which were related to the histocompatibility (H-2) type of the strains. Immunization with a single high dose (100 micrograms) of antigen failed to demonstrate these differences.

Infections and autoimmunity: a panorama.

For more than 2,000 years, it was thought that malignant spirits caused diseases. By the end of nineteenth century, these beliefs were displaced by more modern concepts of disease, namely, the formulation of the "germ theory," which asserted that bacteria or other microorganisms caused disease. With the emergence of chronic degenerative and of autoimmune diseases in the last century, the causative role of microorganisms has been intensely debated; however, no clear explanatory models have been achieved. In this review, we examine the current available literature regarding the relationships between infections and 16 autoimmune diseases. We critically analyzed clinical, serological, and molecular associations, and reviewed experimental models of induction of and, alternatively, protection from autoimmune diseases by infection. After reviewing several studies and reports, a clinical and experimental pattern emerges: Chronic and multiple infections with viruses, such as Epstein-Barr virus and cytomegalovirus, and bacteria, such as H. pylori, may, in susceptible individuals, play a role in the evolvement of autoimmune diseases. As the vast majority of infections pertain to our resident microbiota and endogenous retroviruses and healthy carriage of infections is the rule, we propose to focus on understanding the mechanisms of this healthy carrier state and what changes its configurations to infectious syndromes, to the restoration of health, or to the sustaining of illness into a chronic state and/or autoimmune disease. It seems that in the development of this healthy carriage state, the infection or colonization in early stages of ontogenesis with key microorganisms, also called 'old friends' (lactobacilli, bifidobacteria among others), are important for the healthy living and for the protection from infectious and autoimmune syndromes.

Evolution and conservation of immunological activity.

Paraphrasing what Gregory Bateson says on evolution, we might say that: "Immunology has long been badly taught. In particular, students--and even professional immunologists--acquire theories of immunological activity without any deep understanding of what problems these theories attempt to solve."

Immunoglobulin production is impaired in protein-deprived mice and can be restored by dietary protein supplementation.

Most contacts with food protein and microbiota antigens occur at the level of the gut mucosa. In animal models where this natural stimulation is absent, such as germ-free and antigen-free mice, the gut-associated lymphoid tissue (GALT) and systemic immunological activities are underdeveloped. We have shown that food proteins play a critical role in the full development of the immune system. C57BL/6 mice weaned to a diet in which intact proteins are replaced by equivalent amounts of amino acids (Aa diet) have a poorly developed GALT as well as low levels of serum immunoglobulins (total Ig, IgG, and IgA, but not IgM). In the present study, we evaluated whether the introduction of a protein-containing diet in 10 adult Aa-fed C57BL/6 mice could restore their immunoglobulin levels and whether this recovery was dependent on the amount of dietary protein. After the introduction of a casein-containing diet, Aa-fed mice presented a fast recovery (after 7 days) of secretory IgA (from 0.33 to 0.75 mg/mL, while in casein-fed mice this value was 0.81 mg/mL) and serum immunoglobulin levels (from 5.39 to 10.25 mg/mL of total Ig). Five percent dietary casein was enough to promote the restoration of secretory IgA and serum immunoglobulin levels to a normal range after 30 days feeding casein diet (as in casein-fed mice--15% by weight of diet). These data suggest that the defect detected in the immunoglobulin levels was a reversible result of the absence of food proteins as an antigenic stimulus. They also indicate that the deleterious consequences of malnutrition at an early age for some immune functions may be restored by therapeutic intervention later in life.

Aging affects oral tolerance induction but not its maintenance in mice.

B6D2F1 mice, which are very susceptible to tolerance induction by a single gavage with 20 mg of ovalbumin (Ova) at age 8 weeks, become less susceptible at age 25 weeks and totally refractory at age 70 weeks. However, 70-week-old mice may be rendered tolerant by repeated ingestion of Ova. Mice orally exposed to Ova at age 8 weeks remain tolerant at age 70 weeks. The isotypic pattern of anti-Ova antibodies formed by orally-tolerant and normal mice after immunization is similar and all isotypes are equally suppressed by oral tolerance. In old mice, oral exposures to Ova alone triggered an early transient antibody response; some of these responding animals were, nevertheless, tolerant to subsequent parenteral injection of Ova in adjuvant.

Anti-gamma delta T cell antibody blocks the induction and maintenance of oral tolerance to ovalbumin in mice.

The oral administration of antigens is one of the means of inducing tolerance in adult mammals. In this report, the role of gamma delta T cells in the induction and maintenance of orally-induced tolerance to ovalbumin was investigated. The injection of a monoclonal anti-gamma delta T cell monoclonal antibody blocked the induction of oral tolerance, because the secondary immune responses to ovalbumin in these animals were comparable to the corresponding responses in ovalbumin-immunized control mice. Furthermore, depletion of gamma delta T cells either in vivo or in vitro abolished already established oral-tolerance. The fact that the state of tolerance could be adoptively transferred to naive recipients by CD3+ alpha beta- gamma delta + spleen cells from tolerant mice. These results suggest that systemic oral tolerance is induced and actively maintained by mechanisms involving gamma delta T cells.

Specific responses to two unrelated antigens in mice made orally tolerant to one of them.

BDF1 and B10 mice, either normal or exclusively fed egg white diluted in water, were immunized with ovalbumin (OVA), sheep erythrocytes (SRBC) or OVA + SRBC. BDF1, but not B10, mice became tolerant to OVA and presented a 100% increase in the total number of immunoglobulin-secreting cells in the spleen. Immunization with OVA + SRBC markedly increased the response to OVA, except in tolerant BDF1 mice, and had no effect on the anti-SRBC PFC response, except in normal (non-tolerant) BDF1 mice.

Oral tolerance induction with altered forms of ovalbumin.

As a T cell-dependent phenomenon, oral tolerance is not expected to depend necessarily on native configuration of antigens. We investigated the induction of oral tolerance with modified ovalbumin (Ova). Oral administration of heat-denatured (HD-Ova) and cyanogen bromide-degraded ovalbumin was less effective than native Ova in inducing oral tolerance in B6D2F1 mice. HD-Ova was effective in suppressing delayed-type hypersensitivity (DTH) reactions but did not suppress specific antibody formation. Injection of Ova directly into the stomach, but not into the ileum or cecum, suppressed subsequent immunization to DTH reactions. Gavage with protease inhibitors (aprotinin or ovomucoid) before gavage with Ova was ineffective in blocking tolerance induction. Treatment with hydroxyurea to destroy cycling cells 24 h before gavage with Ova blocked oral tolerance induction and also the possibility to passively transfer tolerance to naive recipients with the serum of mice gavaged with Ova 1 h before. The implications of these findings about oral tolerance induction are discussed.

Ethanol-induced colitis prevents oral tolerance induction in mice.

The gut mucosa is a major site of contact with antigens from food and microbiota. Usually, these daily contacts with natural antigens do not result in inflammatory reactions; instead they result in a state of systemic hyporesponsiveness named oral tolerance. Inflammatory bowel diseases (IBD) are associated with the breakdown of the immunoregulatory mechanisms that maintain oral tolerance. Several animal models of IBD/colitis are available. In mice, these include targeted disruptions of the genes encoding cytokines, T cell subsets or signaling proteins. Colitis can also be induced by intrarectal administration of chemical substances such as 2,4,6-trinitrobenzene sulfonic acid in 50% ethanol. We report here a novel model of colitis induced by intrarectal administration of 50% ethanol alone. Ethanol-treated mice develop an inflammatory reaction in the colon characterized by an intense inflammatory infiltrate in the mucosa and submucosa of the large intestine. They also present up-regulation of both interferon gamma (IFN-gamma) and interleukin-4 (IL-4) production by cecal lymph node and splenic cells. These results suggest a mixed type of inflammation as the substrate of the colitis. Interestingly, cells from mesenteric lymph nodes of ethanol-treated mice present an increase in IFN-gamma production and a decrease in IL-4 production indicating that the cytokine balance is altered throughout the gut mucosa. Moreover, induction of oral tolerance to ovalbumin is abolished in these animals, strongly suggesting that ethanol-induced colitis interferes with immunoregulatory mechanisms in the intestinal mucosa. This novel model of colitis resembles human IBD. It is easy to reproduce and may help us to understand the mechanisms involved in IBD pathogenesis.

Local anaphylactic reactions to the penetration of cercariae of Schistosoma mansoni.

1. In order to study local tissue anaphylactic responses to infection with Schistosoma mansoni cercariae, mice of three different strains (C57BL/10J, Balb/cJ and CBA/J) were infected by subcutaneous injection of 15 to 20 cercariae. Eleven to 16 weeks later the animals were reinfected through one ear with 250 to 350 cercariae. 2. Throughout the infection period, the histamine content of the ears increased up to 150% of control values. Upon reinfection, the penetration of cercariae through the ear reduced its histamine content to near normal values. 3. Reinfection causes inflammation as judged by a 1.5 to 2.3-fold increase in the amounts of plasma leaking through the ear vessels as measured by leakage of Evans blue dye. 4. These results suggest that the local inflammatory reaction mediated by mast cells is important in the resistance of mice to reinfection with S. mansoni.

Interruption of recently induced immune responses by oral administration of antigen.

Interest in oral tolerance has been renewed in the last few years as a possibility of intervention in human autoimmune diseases. An obstacle in this direction is that, although easily induced in animals virgin of contact with the antigen, oral tolerance becomes hard to induce in previously immunized animals. The present results show that there is an early period after primary immunization in which prolonged oral exposure to the antigen may arrest ongoing immune responses. Beyond this period, oral exposures to the antigen become ineffective and may actually boost immune responses. The end of the susceptible period coincides with the emergence of free specific antibodies in serum. However, the previous administration of purified anti-ovalbumin antibodies (40 micrograms) was unable to block the induction of oral tolerance to ovalbumin in normal mice.

Immunological induction of flavor aversion in mice.

1. Young adult BALB/c and B6D2F1 mice of both sexes (20 +/- 2 g) immunized ip with 2 doses of 10 micrograms ovalbumin (Ova), but not with 2 doses of 10 micrograms bovine gammaglobulins (BGG), show aversion to the ingestion of sweetened egg white or crystallized Ova solutions which are avidly ingested by normal mice. In 24 h, normal mice or mice immunized with BGG ingested, respectively, 340 +/- 80 and 265 +/- 56 mg of sweetened egg white per gram of body weight (mg/gbw); in the same period, Ova-immunized mice ingested less than one tenth these amounts (18 +/- 5 mg/gbw). ELISA-titers of anti-Ova and anti-BGG antibodies in immune mice were of similar magnitude. 2. Aversion arises coincidentally with the emergence of anti-ovalbumin antibodies in serum in the primary response, 14 days after primary immunization. 3. Previous induction of oral tolerance to ovalbumin by a single gavage with 20 mg Ova 7 days before primary ip immunization, which blocks the increase of specific antibodies in serum, also blocks the development of the aversive phenomenon. 4. Aversion was induced to 1 mg/ml but not 0.1 mg/ml sweetened crystallized ovalbumin solutions and was already noticeable 2 h after exposure of immunized mice to sweetened egg white solutions. 5. We conclude that, at least in experimental situations, immunological factors may be of decisive importance in diet selection.

Evidence for the participation of mast cells in the innate resistance of mice to Schistosoma mansoni: effects on in vivo treatment with the ionophore 48-80.

1. Evidence is presented for the participation of mast cells in the resistance of mice to infection by Schistosoma mansoni. 2. Intravenous injection of 1 micrograms/g body weight of the ionophore 48-80, a potent mast cell degranulator, significantly reduced (42-62%) the histamine content of the ear tissue of normal mice and either increased or decreased resistance to parasite penetration and infection depending on whether the injection of the ionophore was 5 min or 2 days before cercarial penetration. 3. When 48-80 was injected 5 min before the beginning of cercarial penetration, the number of parasites recovered from ear tissue 2 days later or from the portal system 30 days later was significantly reduced (39-71% and 27-40%, respectively) in relation to untreated controls. This resistance caused by 48-80 was blocked when mice were simultaneously treated with cyproheptadine, an antagonist of vasoactive amines. 4. In contrast, when 48-80 was administered 2 days before the beginning of cercarial penetration, the number of parasites retrieved from ear tissue 2 days later or from the portal system 30 days later was 32-64% and 28-30% larger, respectively, in relation to untreated controls. 5. These findings indicate that the local inflammatory reaction mediated by mast cells is important in the resistance of mice to infection with S. mansoni.

Indirect effects of oral tolerance to ovalbumin interfere with the immune responses triggered by Schistosoma mansoni eggs.

The objective of the present study was to investigate whether the injection of a tolerated protein (indirect effects) affects the formation of granulomas around Schistosoma mansoni eggs trapped in the lungs after intravenous (iv) injection into normal (noninfected) C57BL/6 mice (6 animals per group). To induce oral tolerance to chicken egg ovalbumin a 1/5 dilution of egg white in water was offered ad libitum in a drinking bottle for 3 days. Control mice received water. After 7 days, control and experimental animals were injected iv with 2,000 S. mansoni eggs through a tail vein. In some mice of both groups the iv injection of eggs was immediately followed by intraperitoneal (ip) immunization with 10 micro g of dinitrophenylated conjugates of ovalbumin (DNP-Ova) emulsified in complete Freund's adjuvant (CFA) or only CFA; 18 days later, mice were bled and killed by ether inhalation. The lungs were fixed in formalin and embedded in paraffin. Serial sections of 5 m were stained with Giemsa, Gomori's silver reticulin and Sirius red (pH 10.2). Granuloma diameters were measured in histological sections previously stained with Gomori's reticulin. Anti-DNP and anti-soluble egg antigen (SEA) antibodies were analyzed by ELISA. In mice orally tolerant to ovalbumin the concomitant ip injection of DNP-Ova resulted in significantly lower anti-SEA antibodies (ELISA*: 1395 +/- 352 in non-tolerant and 462 +/- 146 in tolerant mice) and affected granuloma formation around eggs, significantly decreasing granuloma size (area: 22,260 +/- 2478 to 12,993 +/- 3242 m ). Active mechanisms triggered by injection of tolerated antigen (ovalbumin) reduce granuloma formation.

Stabilization of serum antibody responses triggered by initial mucosal contact with the antigen independently of oral tolerance induction.

Initial contacts with a T-dependent antigen by mucosal routes may result in oral tolerance, defined as the inhibition of specific antibody formation after subsequent parenteral immunizations with the same antigen. We describe here an additional and permanent consequence of these initial contacts, namely, the blockade of secondary-type responsiveness to subsequent parenteral contacts with the antigen. When repeatedly boosted ip with small doses (3 microg) of ovalbumin (OVA) (or lysozyme), primed B6D2F1 mice showed progressively higher antibody responses. In contrast, mice primed after a single oral exposure to the antigen, although repeatedly boosted, maintained their secondary antibody titers on a level which was inversely proportional to the dose of antigen in the oral pretreatment. This phenomenon also occurred in situations in which oral tolerance was not induced. For example, senile 70-week-old B6D2F1 mice pretreated with a single gavage of 20 mg OVA did not become tolerant, i.e., they formed the same secondary levels of anti-OVA antibodies as non-pretreated mice. However, after 4 weekly challenges with 3 microg OVA ip, orally pretreated mice maintained the same anti-OVA serum levels, whereas the levels of control mice increased sequentially. This "stabilizing" effect of mucosal exposure was dose dependent, occurred with different proteins and was triggered by single or multiple oral or nasal exposures to the antigen.

Hydroxyurea before oral antigen blocks the induction of oral tolerance.

1. Treatment with hydroxyurea (HU, 1 mg/g ip, 2 doses applied 7 h apart) eliminates the majority of cells undergoing mitosis (cycling cells) without affecting non-cycling cells. Oral tolerance, induced by a single gavage with 20 mg of ovalbumin, results in a drastic inhibition of anti-Ova antibody responses in young adult mice. Oral tolerance is actively maintained by the presence of specific suppressor T cells which may adoptively transfer the tolerance to naive syngeneic recipients. Under the clonal selection hypothesis, the induction of oral tolerance should be blocked by HU treatment applied soon after oral exposure to the antigen by the elimination of specific clones of lymphocytes activated by tolerogenic presentation of the antigen. 2. However, treatment with HU initiated 3, 6 or 24 h after oral exposure to ovalbumin had no effect on the induction of oral tolerance in B6D2F1 mice. However, treatment with HU 24 h before antigen exposure, totally blocked the induction of tolerance. Treatment with HU 72 h before ovalbumin had no effect. 3. In animals treated with HU 24 h before, the adoptive transfer of normal thymus, bone marrow or spleen cells partially restored the susceptibility to the induction of oral tolerance. 4. The results suggest that cycling cells, which may be totally regenerated within 72 h after treatment with HU, and are present in normal thymus, bone marrow and spleen, are crucially important for the induction of oral tolerance.

Systemic immunization of mature mice by the oral route.

1. Mice of several strains which are susceptible to the induction of oral tolerance by a single gavage with 20 mg of ovalbumin (Ova) when young adults (7-8 weeks old) become less susceptible or refractory to tolerance induction when mature (20-40 weeks old). The antibody-forming capacity of these mature animals remains invariant compared to young adults (8-10 weeks old). 2. Mature mice of several strains display significant serum antibody responses to 3 gavages (days 0, 7 and 28) with Ova; as assessed by ELISA titers, these responses are similar in magnitude to those elicited by standard ip immunization with small doses of Ova. 3. In mature H-III mice, gavages on days 0, 7 and 28 induced significant antibody formation. On the contrary, the ingestion of the same amounts of Ova on days 0, 7 and 28 induced oral tolerance. Concomitant gavage with saline on the days of Ova ingestion failed to inhibit tolerance induction. 4. In H-III mice displaying circulating antibodies induced by repeated gavage with Ova, additional ip injections of Ova failed to increase the antibody titers.