Surface phenotype of T cells producing growth of mucosal mast cells in normal rat bone marrow culture.

We have previously shown that lymphocytes from Nippostrongylus brasiliensis infected rats, when stimulated with antigen or concanavalin A (Con A) release factors which are comparable with murine IL-3. On addition of these factors to rat bone marrow cultures, mast cells with the morphological and biochemical properties of mucosal mast cells (MMC) proliferate and mature. Here, we use this system, along with monoclonal antibodies against rat T cells and the fluorescence-activated cell sorter (FACS), to isolate the subset of T cells responsible for the production of this MMC growth factor. Lymphocytes from N. brasiliensis infected rats were separated on the FACs into populations with and without the antigens defined by OX19, W3/25 and OX8 monoclonal antibodies; these antibodies label all T cells, T-helper cells and T-cytotoxic/suppressor cells, respectively. The resultant subsets were cultured in vitro with Con A. The supernatants were tested for the ability to induce MMC growth and differentiation in liquid cultures of normal rat bone marrow. The phenotype of the T cells producing this factor was established as being OX19+, W3/25+ and OX8-.

Immunoregulation of IgE responses: the role of the gut in perspective.

In adult rats and mice of most strains the experience of antigen whether by mouth or by injection usually leads to a diminished rather than a heightened IgE response to a subsequent exposure. This is because these animals have a highly developed capacity to suppress the production of IgE by immunoregulatory mechanisms which can be activated by the administration of even minute quantities of antigen. The situation in young animals is less clear but there are indications that the suppressive mechanisms are less well developed. However, recent experiments in rats have shown that a relatively long lasting suppression of IgE responsiveness occurs in the offspring of immunized mothers and that this effect is mediated by maternal IgG. In this way, the influence of the immunized mother can be seen to compensate for any deficiencies of the developing immunoregulatory mechanisms of the young animal. These experiments were designed to explore ideas about the physiological regulation of IgE responses.

Perinatal influences on IgE responses.

IgE-mediated (atopic) allergy depends on the production of IgE antibodies to normally harmless substances. It is associated with other immunological abnormalities, particularly defects in the T lymphocyte system, and is now generally considered to be a manifestation of IgE-suppressive immunodeficiency. Experiments with adult laboratory animals have revealed the existence of an IgE-selective immunoregulatory system and great efforts are being made to understand the mechanisms by which this system operates, to compensate for, or correct, the deficiency of atopic individuals. However, since allergy is often a problem of childhood one might reasonably ask if experiments with adult animals are a wholly appropriate approach to the problem. Rats were used to explore factors influencing the development of IgE regulation during early life, when the immunological apparatus differs intrinsically from that of mature animals. The results of these experiments, which have shown profound IgE-suppressive effects of maternal antibody and of neonatally ingested antigen, are reviewed with the aim of stimulating research on this topic in man.

The development of IgE-suppressive immunocompetence in young animals: influence of exposure to antigen in the presence or absence of maternal immunity.

Previous studies recognized a prolonged suppression of egg-albumin (EA)-specific IgE responsiveness in the progeny of immunized female rats, and showed that an identical effect was produced by the administration of small amounts of specific IgG during the first few days of life. Both manipulations also elevated the primary IgG response to a subsequent immunization but with less consistency. We have now investigated the effects on the progeny of varying the type of maternal response by immunizing with antigen given with or without adjuvant. Mothers immunized with EA without adjuvant (by mouth or parenterally) in whom both IgE and IgG responses are thereby suppressed do not influence the antibody-responsiveness of their progeny. By contrast, mothers immunized with EA in adjuvant, a procedure which suppresses IgE but enhances IgG responsiveness to the antigen, transfer both effects to their offspring. In this way, both IgE-suppression and IgG-enhancement are seen to correlate with the transmission of maternal IgG. EA administered either by mouth or parenterally in the period up to 6 weeks after birth, suppresses both IgE and IgG responses to subsequent specific challenges in a dose-dependent manner. The results of feeding antigen to the progeny of (IgG-transmitting) immune mothers indicated that passive and active immunity in the young rat, although both suppressing IgE-responsiveness, do not have additive depressive effects.

In vitro studies on mast cell proliferation in N. brasiliensis infection.

We have previously shown that mast cells with the morphological and biochemical properties of mucosal mast cells (MMC) proliferate and mature in rat bone marrow cultures stimulated with factors from antigen or mitogen-activated T lymphocytes. Here we have used this system to explore the MMC hyperplasia which occurs in infections with gastrointestinal nematode parasites. Lymphocytes producing MMC-growth factor were present from day 10 onwards in N. brasiliensis-infected rats and mesenteric lymph nodes (MLN) were the major source of activated lymphocytes. When different tissues of normal rats were cultured in the presence of conditioned medium by far the greatest proliferation of MMC occurred in bone marrow, indicating an origin of MMC from haemopoietic precursors. Cultures of infected rat bone marrow yielded considerably greater numbers of MMC than cultures of normal rat bone marrow and experiments using semisolid culture media indicated that N. brasiliensis infection causes an increase in the frequency of MMC progenitors in the bone marrow. A scheme is put forward for the sequence of events occurring in vivo based on the results of these and other published experiments. The reasons for the restricted in vivo localization of MMC to the mucous membranes and associated lymph nodes is discussed. Finally we give the results of microspectrophotometric analysis which has shown that the cultured mast cell contain a non-heparin proteoglycan, thus adding a further feature to the list of MMC-like properties of these cells.

Mucosal mast cells in vivo and in vitro.

There has recently been a flurry on the mast cell front caused, not least, by the publication of articles about the IL3-induced growth of mast cells in vitro from haemopoietic tissue. This has left bystanders, and one suspects not a few participants, confused about some of the issues involved. The reason for the confusion lies in the participation of disparate specialities - such as experimental haematology and immunoparasitology - among which communication has not been traditional: however, the revelations raise specific questions which demand an interdisciplinary approach. Thus the evidence is strong that the mast cells derived from cultured haemopoietic tissues are of a special type hitherto called atypical, intestinal or mucosal mast cells (MMC) which are known to occur in profusion in mucous membranes of helminth-infected animals. In this article Ellen Jarrett and David Haig link information about these cells obtained from both in-vivo and in-vitro experiments.

IgE production in rat fascioliasis.

F. hepatica infection of rats caused a prolonged elevation of serum total IgE reflecting the continued presence of live worms in the host. Infection with 40 metacercariae stimulated higher total IgE levels than infection with 20 metacercariae. The parasite specific IgE response was biphasic, the first peak coinciding with the migratory phase in the liver parenchyma and the second with the establishment of flukes in the bile ducts.

Stimulation of mucosal mast cell growth in normal and nude rat bone marrow cultures.

Mast cells with the morphological and biochemical properties of mucosal mast cells (MMC) appear and proliferate to form the predominant cell type in rat bone marrow cultures stimulated with factors from antigen- or mitogen-activated lymphocytes. Conditioned media causing a selective proliferation of MMC were derived from mesenteric lymph node cells of Nippostrongylus brasiliensis-infected rats restimulated in vitro with specific antigen or from normal or infected rat mesenteric lymph node cells stimulated with concanavalin A. MMC growth factor is not produced by T-cell-depleted mesenteric lymph node cells or by the mesenteric lymph node cells of athymic rats. By contrast, MMC precursors are present in the bone marrow of athymic rats and are normally receptive to the growth factor produced by the lymphocytes of thymus-intact rats. The thymus dependence of MMC hyperplasia is thus based on the requirement of a thymus-independent precursor for a T-cell-derived growth promoter.

IgE suppression by maternal IgG.

Rats born of egg albumin immunized mothers have a diminished capacity to produce IgE antibody to egg albumin persisting for at least 13-14 weeks after birth. At the same time the primary IgG response to the antigen is usually enhanced. Previous studies indicated that these effects were mediated by factors transferred in maternal milk. The phenomenon can be duplicated by the administration of small quantities of immune serum to rats during the first 3 weeks of life. The active component of immune serum is shown to be specific antibody. Suckling rats acquire egg-albumin-specific IgG from the immune mother via the milk. Their serum level approaches that of the mother by 20 days but declines rapidly after weaning to become undetectable by 6-8 weeks. As maternal influence on the immune responsiveness of the offspring persists for several weeks beyond this time, it is unlikely that the mechanism involves a simple blocking by circulating antibody of the access of antigen to cellular receptors. Alternative mechanisms are briefly discussed. Attention is drawn to the possibility that the suppression of IgE antibody responsiveness by maternal IgG may represent a physiological regulatory process with a role in inhibiting the development of infantile allergies.

Generation of mucosal mast cells is stimulated in vitro by factors derived from T cells of helminth-infected rats.

The connective tissue of rats, and several other species of mammals, contains two distinct types of mast cells that differ in morphology, histochemical staining properties and location1. One type, frequently called the normal connective tissue mast cell, can be obtained in nearly homogeneous preparation from a mixed cell population in the peritoneal cavity and forms the basis of our knowledge of mast cells. The other type is referred to as the mucosal mast cell because in normal rats it has been observed only in mucosal tissue. Infection with helminth parasites induces an exteNsive accumulation of mast cells and eosinophils in the tissues, and parasites of mucous surfaces, in particular, stimulate a rapid hyperplasia of mucosal mast cells. However, the origin of mucosal mast cells, and their relationship to the connective tissue mast cells is uncertain. We now slow that lymphocytes of helminth-infected rats, on in vitro stimulation with specific antigen, release factors causing pronounced mucosal mastocytosis in normal rat bone marrow cultures.

Adoptive transfer of total and parasite-specific IgE responses in rats infected with Nippostrongylus brasiliensis.

Infection of rats with Nippostrongylus brasiliensis has both a parasite-specific and non-specific IgE stimulating effect. Both these responses can be adoptively transferred with thoracic duct lymphocytes (TDL) from infected rats. The character of the IgE response in the recipient rats was related to the stage after infection of the cell donors. TDL from hyperimmune rats adoptively transferred high serum titres of parasite-specific IgE to infected recipient rats and substantially increased the levels of total IgE. However, adoptive immunization with TDL from donors infected 10 days previously did not stimulate parasite-specific IgE and only slightly increased total IgE levels. After cell fractionation the sIg- cells from day 10 TDL increased the level of total IgE but not parasite-specific IgE whereas sIg- cells from hyperimmune TDL did not induce any IgE response unless given with sIg+ cells. The possible reasons for this are discussed.

Regulation of ongoing IgE antibody responses with minute doses antigen.

Repeated challenge of egg albumin-immunized Hooded Lister rats with a tiny amount of antigen presents a stimulus not only to effector cells for an IgE response but also to a related class-selective suppressive mechanism which eventually becomes predominant. The results obtained suggest that IgE responsiveness is abrogated by exposure to an antigen dose which is several orders of magnitude lower than required for the regulation of antibody of other classes.

Adjuvants in the induction and enhancement of rat IgE responses.

In Hooded Lister rats the primary IgE antibody response induced by immunization with antigen and adjuvant may be enhanced either specifically by a further dose of antigen (booster response) or non-specifically by infection with helminth parasites (potentiated response). The initial immunizing technique can influence the occurrence and level of these enhanced responses and here we describe the effect of using different adjuvants in the priming event. Although the level of the primary response was broadly similar following immunization with egg-albumin and the adjuvants Bordetella pertussis aluminium hydroxide or Freund's complete adjuvant, the booster response was inhibited and the potentiated response intensified in animals immunized with the latter two adjuvants. A significant IgE booster response could only be obtained if B. pertussis had been used in the initial immunization. When aluminium hydroxide was adsorbed to B. pertussis it was found to have the same inhibitory effect on subsequent booster responses as when it was adsorbed to antigen. These results are discussed in relation to the intricacies of IgE production in the present model and to more general mechanisms of adjuvant action.

A radioimmunoassay for evaluation of the IgE and IgG antibody responses in the rat.

An assay, the paper radioallergosorbent test (PRAST), for the measurement of specific serum IgE antibody in the rat is described in detail. This assay has been used, in conjunction with a modified PRAST for the determination of relative specific serum IgG antibody and the PRIST assay for total serum IgE [13], to measure specific IgE and IgG and total IgE immune responses in normal parasite infected rats immunized using various protocols. The results indicate that there is a relationship between the basic IgE level and the immune response, i.e. a rat strain with a low constitutive IgE level demonstrates a weak response whereas a high level strain reacts strongly. When PRAST and passive cutaneous anaphylaxis (PCA) were compared, using standardized IgE antibody containing sera, the results were in good agreement. However, PRAST is the preferable assay as it shows less intrinsic variation, is more sensitive than PCA, and is not influenced by high serum IgE levels in the recipient animal.

Stimuli for the production and control of IgE in rats.

In Hooded Lister rats IgE responses may be induced by administration of antigen together with one of a number of adjuvants. The primary IgE response may subsequently be enhanced either specifically by a further exposure to antigen (booster response) or non-specifically by infection with helminth parasites (potentiated response). In the latter case the enhanced response is associated with a great increase in total serum IgE. The primary response itself is not significantly influenced by variations in the general theme of conventional immunization, including dose or route of administration of antigen, or the nature of the adjuvant employed. The booster response however is inhibited, a) in rats primed with a 'large' (e.g. greater than 100 microgram EA) dose of antigen and B. pertussis, and b) rats primed with any dose of antigen given in Al(OH)3 or CFA, and c) following repeated booster doses of soluble (i.e. unadjuvanted antigen even at a dosage of a few picogrammes. It is thought that each of the stimuli generate antigen specific suppressor T cells. Live worm parasites selectively, but non-specifically, stimulate heterologous antigen primed IgE responses. The evidence suggests that it may be this non-specific IgE stimulating effect rather than the parasite specific IgE response per se which leads to the great elevation of total serum IgE. Other immunoglobulin classes are not elevated in the same way. The potentiated IgE response is not susceptible to the suppressive influence generated by previous administration of large or repeated doses of the heterologous antigen. On the other hand, a parasite specific regulatory mechanism acts to prevent repotentiation of the heterologous (but not the parasite specific or total IgE) responses following reinfection. These results are discussed in relation to the work of others in rats and other species.

Serum immunoglobulin levels in N. brasiliensis infection.

We have measured the levels of IgG1, IgG2a, IgG2c, IgA and IgM in the serum of normal rats, and at various times after infection or re-infection with Nippostrongylus brasiliensis, in order to compare these responses with previously measured IgE levels. The results we have to report are that while the levels of the various immunoglobulin classes or subclasses are elevated to a greater or lesser extent, the increments are modest by comparison with the great elevation of total IgE.

Activation of IgE regulatory mechanisms by transmucosal absorption of antigen.

The development of immediate hypersensitivities depends essentially on the production of IgE antibodies--usually to common environmental antigens. It has been suggested that atopic individuals produce IgE antibodies as a result of overstimulation with antigen and that this occurs through IgA deficiency which by default allows the absorption via the mucosae of abnormally large amounts of antigen. However, work with laboratory animals indicates a mechanism which is the antithesis of the overstimulation concept: that quantities of antigen sufficiently large to activate IgE immunoregulatory mechanisms, particularly suppressor T cells, are normally absorbed across the mucosae and that, where other conditions for the activation of these cells are appropriate, inhibition rather than stimulation of IgE responses results. In this way allergies arise through a defect of one or more components of the immunoregulatory mechanism. The fact that atopic individuals do not become allergic to all ubiquitous antigens and that they may be hyposensitised is evidence that the defect is relative rather than absolute.