Induction of an auto-anti-IgE response in rats I. Effects on serum IgE concentrations

With a view to specifically suppressing the IgE isotype, rats of high (BN) and low (PVG.RT1u) IgE-responding phenotypes were immunized with a highly purified rat IgE myeloma (IR2) in an attempt to induce an anto-anti-IgE response. Rat IgE antibodies against epsilon determinants were detected in the serum of IR2-immunized animals using a solid-phase (plate) radioimmunoassay. The auto-anti-IgE antibodies detected were found to bind to IR2, to a second rat IgE myeloma (IR162) and to mouse monoclonal IgE but not rat IgG. The specificity of the anti-epsilon binding was shown by inhibition studies. The raising of an auto-anti-IgE response in PVG.RT1u rats severely depleted the serum level of circulating IgE for at least 8 weeks. In BN rats, immunization with IR2 caused marked fluctuations in serum IgE levels. The rats in both strains remained healthy throughout the experiment. The rate and route of IgE break down was not altered in anti-IgE-producing rats. The relevance of the present model in understanding and possibly controlling allergic disorders is considered.     

Induction of an auto-anti-IgE response in rats. IV. Effects on mast cell degranulation.

Induction of an auto-anti-IgE (auto-aIgE) response in the rat inhibits both total and specific IgE production and alters the distribution of mast cell (MC) subpopulations identified by differential Alcian blue/safranin staining. We have extended these observations by characterizing the auto-aIgE antibodies and determining their effects on MC degranulation in vitro and in vivo. An auto-aIgE response was induced in bacillus Calmette-Guérin (BCG)-primed rats by injecting a conjugate of highly purified rat IgE myeloma (IR2) coupled to tuberculin-derived purified protein derivative (PPD). Anti-IgE autoantibodies were almost exclusively IgG2a. The intradermal injection of auto-aIgE into untreated rats induced local MC degranulation as shown by a strong immediate skin response. Histologically there was evidence of significant degranulation of safranin staining connective tissue MC (SMC) in the skin but not of the Alcian blue staining MC (ABMC) in the sub-epidermal region. The induced degranulation was epsilon-chain specific; immunopurified anti-idiotypic antibodies raised to the IgE IR2 myeloma had no MC degranulating activity. When administered locally, auto-aIgE inhibited a subsequent passive cutaneous anaphylaxis (PCA) response elicited by anti-ovalbumin IgE. In addition, the PCA response was significantly decreased in animals with an ongoing auto-aIgE response. Immunopurified auto-aIgE also induced histamine release in vitro from rat peritoneal MC. These results are discussed in the context of naturally occurring autoantibodies to IgE present in patients with allergic disease.     

Induction of an auto-anti-IgE response in rats. III. Inhibition of a specific IgE response.

An auto-anti-IgE response was induced in conventional (PVG-RT1U and high IgE-producing (BN) rat strains by immunization with a highly purified rat IgE myeloma IR2. Earlier work established that total serum IgE levels were decreased by this procedure (Marshall & Bell, 1985) but only in the PVG-RT1U strain. IR2-immunized rats were tested for their ability to produce a specific IgE response to ovalbumin (OVA). The primary anti-OVA IgE response was inhibited by 60-75% in both rat strains, regardless of whether the total serum levels of IgE were reduced. The secondary IgE response to OVA was also inhibited in anti-IgE-producing animals but not in rats primed with OVA before anti-IgE induction. The inhibition of the anti-OVA response was isotype specific; the IgG response to OVA was unaffected. These studies may help elucidate the regulatory role played by naturally occurring anti-IgE antibodies found particularly in atopic individuals.     

In vivo arming of cutaneous mast cell receptors by IgE released from macrophages

Intracutaneous injection of purified peritoneal macrophages harvested from ovalbumin (OVA)-hypersensitive high-IgE-responder BN rats into naive animals sensitised the injection sites for subsequent OVA-specific passive cutaneous anaphylaxis (PCA) reactions. The underlying mechanism(s) were investigated using a macrophage cell line (WEHI 265.1), which exhibited comparable sensitising activity in rat or mouse skin, after initial pulsing in vitro with antiserum rich in OVA-specific IgE. Transfer of OVA-hypersensitivity by the cell line (1) was IgE-dependent and did not occur when the cells were pre-exposed to antiserum containing OVA-specific IgG alone, (2) was blockable by saturation of cell surface receptors in the recipient with myeloma IgE (but not myeloma IgG), and (3) did not occur in mast cell-deficient mice carrying the W/Wv mutation, in contrast to their normal heterozygous littermates which developed marked OVA-hypersensitivity at the injection site. These results are consistent with arming of IgE-receptors on cutaneous mast cells by IgE antibody released from macrophages, and hint at a possible role for phagocytes in amplifying IgE-mediated reactions in tissues.     

Accelerated elimination of N. brasiliensis from the small intestine after auto-anti-IgE induction.

Immunization of rats with a purified IgE myeloma (IR2) induced an auto-anti-IgE response. Such treatment inhibited total IgE levels in the serum of conventional IgE-producing rats (Marshall & Bell, 1985) and increased the number of mucosal mast cells (MMC) in the intestine. The present study has investigated the ability of auto-anti-IgE induction to influence the course of a Nippostrongylus brasiliensis infection, to modify IgE synthesis, or to affect the number of MMC in the intestine following infection. Auto-anti-IgE induction was found to have a surprising effect on worm elimination. IR2-immunized rats were able to rid themselves of this nematode with an accelerated tempo--a small but significant effect after primary infection, but a substantial enhancement of worm loss after reinfection. Auto-anti-IgE induction was not able to prevent the typical increase in IgE that accompanies an N. brasiliensis infection, nor did it alter the helminth-induced intestinal mastocytosis. When MMC degranulation was measured by assaying the serum levels of a specific rat mast protease (RMCP II) following secondary infection, the amount of RMCP II released was less in auto-anti-IgE-producing rats. These findings have implications for the importance of IgE, MMC and other cells of inflammation in an anti-parasitic response.     

Anti-IgE-induced histamine release from rat tissues passively sensitizedin vivo with myeloma IgE differs with strain and mast cell source

Four different strains of rats, BDII, E3, LE, and OM/N, each with a low basal serum level of IgE, were examined with respect to anti-IgE- and ConA-induced release of histamine from serosal mast cells and chopped lung tissue. Tests were performed before and three days after i.p. injection of a graded dose of myeloma IgE (IR 162)-containing ascitic fluid. There was a clearcut strain difference in response capacity with respect to ConA- and anti-IgE-induced release of histamine from serosal mast cells before myeloma IgE-injection. Response capacity increased in some but not all strains after myeloma IgE injection; increase in response capacity of serosal mast cells did not correlate to that of chopped lung tissue. Analogous findings were observed when two of the strains, LE and E3, were passively sensitized by i.p. injection with serum containing another myeloma IgE (IR2). These results indicate that differences exist between mast cells of different rat strains, and within strain between mast cells of various tissues in capacity to become sensitized to myeloma IgE.Subsidiary of AB Astra, Sweden.     

Immature peritoneal mast cells in neonatal rats express the CTMC phenotype, as well as functional IgE receptors.

The purpose of this study was to investigate the relationship between the differentiation and maturation of mast cells and the expression of IgE receptors on their surface in neonatal animals in vivo. Another aim was to clarify whether connective tissue mast cells (CTMC) undergo a maturation process involving a transdifferentiation from mucosal mast cells (MMC) during this period of time. Mast-cell phenotypes were studied in terms of the profiles of proteinases and proteoglycan. In 1-week-old rats, the mast-cell granules stained with Alcian blue rather than with safranin (AB+/S-) in the Alcian blue/safranin staining sequence, normally regarded as a property of MMC. However, the AB+/S-stained proteoglycan was degradable by nitrous acid and stained with berberine sulphate, thus indicating that it contained heparin rather than chondroitin sulphate. The mast cells expressed rat mast-cell proteinase (RMCP) I rather than RMCP II, which is normally found in MMC. The mast cells of 1-week-old rats expressed functional IgE receptors, by showing a dose-dependent IgE-mediated histamine release of mast cells. About 70% of the IgE receptors on the mast cells were occupied by IgE. In 2- to 3-week-old rats, there was a progressive increase in mast cells stained with both Alcian blue and safranin or with safranin alone, i.e. they gradually changed towards the staining properties of CTMC (AB-/S+). The expression and the degree of IgE occupancy of the receptors increased in 1- to 3-week-old animals. This was paralleled by an increment in cell size and in the content of heparin, histamine and serotonin in the mast cells. The findings thus indicate that the peritoneal mast cells of neonatal rats express the CTMC phenotype and undergo a maturation process at from 1 to 3 weeks of age, without involving a transdifferentiation from MMC. The maturation of the mast cells is accompanied by an increase in the expression of functional IgE receptors on the cell surface. production was detectable as early as in 1-week-old rats.     

Postnatal maturation of mast cell subpopulations in the rat respiratory tract.

The distribution and enumeration of mast cell subpopulations within the respiratory tract of a high- and low-Ige responder rat strain was determined during postnatal development. Mast cells were identified in adjacent sections by the alcian blue (AB)/safranin (SAF) staining sequence, or using immunoperoxidase to detect the rat mast cell proteinases I (RMCPI) or II (RMCPII). At birth both mucosal mast cells (MMC) and connective tissue mast cells (CTMC) were represented in very low numbers at distinct locations throughout the respiratory tract. The total number of mast cells increased with age. MMC (AB+/RMCPII+ mast cells) were the predominant phenotype in the epithelium and lamina propria of the trachea and the major conducting airways of the lung in all age groups. In contrast, CTMC (AB+/RPMCPI+ and SAF+/RMCPI+ mast cells) predominated in the submucosa of the trachea and major conducting airways as well as in the parenchyma and visceral pleura of the peripheral lung. Both phenotypes co-exist in similar proportions in peribronchial adventitial tissue and adventitia surrounding large blood vessels in neonates as well as adults. In rats the tracheal epithelium is densely populated by MMC from around the time of weaning (3 weeks) and a small but generalized increase in the number of MMC at all sites within the respiratory tract is noted from this time. This increase in MMC frequency in tissue sections with increasing age is mirrored by the levels of circulating serum RMCPII. The number of bone marrow-derived MMC also increased with increasing age prior to weaning, with a significant drop (P less than 0.01) at 4 weeks of age before returning to the peak numbers in 3-week-old rats. The high-IgE responder Brown Norwegian (BN) rat strain constitutively produces significantly more IgE than the low-IgE responder White albino Glaxo (WAG) strain (P less than 0.001) at all ages studied. In contrast, only minor differences between the number and distribution of mast cells in the two strains were observed.     

Compounds that induce autoimmunity in the Brown Norway rat sensitize mast cells for mediator release and interleukin-4 expression

Brown Norway (BN) rats given mercuric chloride (HgCl₂), gold (Au) salts or D-penicillamine develop a T helper 2 (Th2) cell-mediated autoimmune syndrome. The recent observation of tissue injury within 24 h of HgCl₂ treatment suggested the involvement of a non-T cell. We therefore examined the effect of these compounds on rat mast cells in vitro. Incubation of BN rat peritoneal mast cells with HgCl₂ enhanced the release of serotonin in response to IgE cross-linking agents. Mast cells from Lewis rats, a strain not susceptible to the autoimmune syndrome in vivo, were affected to a lesser extent. The effect was observed with purified BN mast cells, suggesting a direct action. Similar effects were seen with D-penicillamine in the presence of copper ions, a combination that produces hydrogen peroxide, and Au. HgCl₂ caused significant induction of interleukin (IL)-4 mRNA in mast cells from BN, but not Lewis rats. The data demonstrate a novel enhancing effect of a number of compounds on mast cell mediator release, and an inducing effect of HgCl₂ on mast cell IL-4 expression. These findings are consistent with our hypotheses that mast cells may contribute to early tissue injury, and also, via production of IL-4, may initiate and/or augment, the Th2 response in the BN rat model of chemical-induced autoimmunity.     

IgG receptors on the mast cell

Antisera to the IgG subclasses, 1, 2a, 2b, and 2c, induced histamine release from mast cells obtained from the peritoneal washings of Lister hooded rats. The maximum responses obtained with anti-IgG₁ and anti-IgG_2a were as great as that for anti-IgE (more than 60% histamine release). Cells from unresponsive Wistar rats which did not secrete appreciable amounts of histamine in response to any of the antisera, produced on active sensitization with ovalbumin a small but significant response on challenge with anti-IgG₁ and anti-IgG_2b as well as with anti-IgE. Passive sensitization with rat myeloma serum of mast cells from the unresponsive rats produced a large response on challenge with anti-IgE but no release to the anti-IgG group 2 subclasses. IgE myeloma serum (11000) neutralized the histamine-releasing activity of anti-IgE serum (87% inhibition) and the antisera to all subclasses of IgG. When the IgE in the myeloma serum was inactivated by heating, the response to the IgG antisera remained completely inhibited except for anti-IgG_2a where some reversal was observed. When purified myeloma IgE (30 g/ml) was used in place of whole serum, marked inhibition (86%) of the response to anti-IgE was obtained leaving the responses to the IgE subclasses unaffected (except for IgG_2a, which was 65% inhibited).     

The strain difference in the effect of mercuric chloride on antigen-triggered serotonin release from rat mast cells is not mediated via interferon-gamma.

Previous work has shown that in vitro exposure of Brown-Norway (BN) rat peritoneal mast cells to mercuric chloride (HgCl2) causes enhancement of subsequent mediator release induced by cross-linking of surface immunoglobulin E (IgE). This enhancing effect is seen significantly less often with peritoneal cells from Lewis rats. In addition HgCl2 has been shown to suppress interferon (IFN)-gamma production by BN but not Lewis splenocytes. Given that IFN-gamma is known to inhibit mediator release by mast cells, we hypothesized that the strain difference in the effect of HgCl2 on mediator release was mediated via a differential effect on IFN-gamma release from T cells in the mixed peritoneal cell population: IFN-gamma release would be suppressed in the case of the BN rat, releasing the mast cells from inhibition and resulting in the enhancing effect of HgCl2. The aim of the study was to test two predictions of this hypothesis. Exposure of BN rat mast cells to IFN-gamma inhibited subsequent antigen-induced mediator release but did not significantly reduce HgCl2-mediated enhancement of this release. Exposure of Lewis rat mast cells to blocking concentrations of anti-IFN-gamma did not reveal any HgCl2-mediated enhancement of mediator release. These observations provide strong evidence against the hypothesis that the differential effects of HgCl2 on BN and Lewis rat mast cells are mediated via IFN-gamma. In addition the results revealed that BN rat mast cells are significantly more sensitive than Lewis rat mast cells to the inhibitory effects of IFN-gamma on antigen-induced mediator release.     

Specific and total IgE responses to antigenic stimuli in Brown-Norway, Lewis and Sprague-Dawley rats.

Kinetics of primary and booster-specific and total IgE responses to distinct antigenic stimuli were studied in two inbred rat strains, Brown-Norway (BN) and Lewis, and one outbred, Sprague-Dawley (SD). The rats were immunized three or four times at intervals varying between 15 and 22 days by subcutaneous injections of 10 microgram ovalbumin, keyhole limpet haemocyanin (KLH) or bovine serum albumin (BSA) mixed with 10 mg aluminium hydroxide gel. IgE antibodies were measured in sera by PCA titres. High responses were obtained in BN rats (PCA titres about 10,000 after booster) and low responses in Lewis and SD rats. Positive booster responses were obtained in the three strains. Peritoneal mast cells collected from the three strains after immunization could degranulate on in vitro addition of specific antigen. In contrast, BN mast cells were bad receptors while Lewis and SD mast cells were good receptors for in vitro passive sensitization by mouse IgE antibodies. Total serum IgE was assayed by an in vitro competitive inhibition bioassay (CIB). The values before immunization were higher in BN (1-4 microgram/ml) than in Lewis (less than 0.25 microgram/ml) or SD rats (0.6 microgram/ml). After immunization, a striking increase could be observed in BN rats (up to 170 microgram/ml). There was no parallel between total IgE and IgE antibody levels at different times after immunization.     

FcγR-dependent apoptosis regulates tissue persistence of mucosal and connective tissue mast cells

Rodent mast cells can be divided into two major subtypes: the mucosal mast cell (MMC) and the connective tissue mast cell (CTMC). A decade-old observation revealed a longer lifespan for CTMC compared with MMC. The precise mechanisms underlying such differential tissue persistence of mast cell subsets have not been described. In this study, we have discovered that mast cells expressing only one receptor, either FcγRIIB or FcγRIIIA, underwent caspase-independent apoptosis in response to IgG immune complex treatment. Lower frequencies of CTMC in mice that lacked either FcγRIIB or FcγRIIIA compared with WT mice were recorded, especially in aged mice. We proposed that this paradigm of FcγR-mediated mast cell apoptosis could account for the more robust persistence of CTMC, which express both FcγRIIB and FcγRIIIA, than MMC, which express only FcγRIIB. Importantly, we reproduced these results using a mast cell engraftment model, which ruled out possible confounding effects of mast cell recruitment or FcγR expression by other cells on mast cell number regulation. In conclusion, our work has uncovered an FcγR-dependent mast cell number regulation paradigm that might provide a mechanistic explanation for the long-observed differential mast cell subset persistence in tissues.     

Complement dependence of histamine release in chronic urticaria.

BACKGROUND: IgG autoantibodies directed to the alpha-subunit of the IgE receptor have been identified in 30% to 45% of patients with chronic urticaria. However, the exact mechanism by which histamine secretion is initiated is uncertain. OBJECTIVE: Histamine release from cutaneous mast cells may occur by cross-linking the IgE receptor or by activation of complement. Our goal is to distinguish these 2 possibilities. METHODS: We incubated human cutaneous mast cells with patient sera, decomplemented sera, or purified patient IgG. The IgG was also added to pooled normal serum or to sera deficient in either C2 or C5, and its ability to activate mast cells was assessed. Mast cells were incubated with human IgE myeloma to saturate alpha-subunits to determine the effect on histamine release. RESULTS: Patient sera released histamine (18.26% +/- 4.39%), but purified IgG from patients (5.5% +/- 4.3%) did not. Addition of the patient IgG to normal sera rendered the sera positive for histamine-releasing activity (18.4% +/- 4.3%), whereas control IgG or patient IgG added to C2- or C5-deficient sera did not release histamine. Histamine release with decomplemented patient sera was also diminished (8.34% +/- 4.3%). Preincubation of mast cells with a C5-blocking peptide decreased histamine release but was not statistically significant; there was a significant decrease after preincubating mast cells with IgE myeloma. CONCLUSION: The degranulation of mast cells by IgG autoantibodies in patients with chronic urticaria requires binding to the IgE receptor and activation of the classical complement cascade. Saturation of the IgE receptor with IgE inhibits such degranulation, presumably by preventing binding of the requisite IgG.     

Binding of human IgE immunoglobulin to rat mast cells. A study by means of three independent techniques.

The binding of human myeloma IgE immunoglobulin on rat mast cells was studied by three independent techniques. A mixed agglutination reaction with anti-IgE-coated Sephadex granules demonstrated that only human IgE-coated rat mast cells were clearly agglutinated. This binding is strong (50% agglutination) in 3 min and progresses for 30 min (95% agglutination). Autoradiographic studies with 125I-labelled human serum proteins demonstrated the selective formation of grains on mast cells incubated with labelled IgE. Upon action of anti-IgE antiserum on IgE-coated rat mast cells, the mast cells released up to 47.5% of their total histamine content in a fluorometric histamine assay. A relationship was established between sensitizing doses of human IgE and histamine release. These results bring evidence for a binding of human IgE on rat mast cells and imply the existence of receptors for this immunoglobulin on mast cell membrane.     

Bovine reaginic antibody. II. Preparation of an anti-reaginic immunoglobulin.

A method of preparing an antiserum to bovine reaginic immunoglobulins is described. This procedure which relies on the ability of bovine reaginic immunoglobulin to attach to rat mast cells, results in an antiserum which upon absorption with rat and bovine materials did not react with rat serum or with bovine IgG1, IgG2, IgM and IgA. The antiserum did, however, react in a gel diffusion assay with PCA positive bovine globulin and a reaginic immunoglobulin-rich fraction of bovine serum. Further, anti-reaginic immunoglobulin activity was shown by binding to and degranulation of rat mast cells previously incubated with PCA positive bovine globulin. The antiserum prevented sensitization of calf skin in a PCA reaction while anti-human IgE did not.     

Role of mouse IgG and IgE homocytotropic antibodies in passive cutaneous anaphylaxis.

The role of the mouse homocytotropic antibodies in passive cutaneous anaphylaxis reaction was investigated. One class of antibody was heat stable, detected at 2 h but not at 48 h after passive transfer, and belonged to a sublcass of mouse IgG. The other was heat labile, detected at 2 h and 48 h after passive transfer, and belonged to the IgE class of mouse immunoglobulins. In the presence of IgG, IgE homocytotropic antibody was not detected early after passive transfer. This was thought to be due to a masking of IgE by IgG antibodies rather than a competition for mast cell surface receptors, since inhibition studies with rat IgE myeloma protein suggested that mouse IgE and IgG1 may have different receptor sites on mast cell surfaces.     

Passive sensitization of mice and rats with IgE antibodies.

The strain-dependent variation in susceptibility to passive sensitization of the skin and peritoneal mast cells with homologous IgE antibodies was observed in inbred strains of mice. Strain 129 showed low susceptibility to passive sensitization and high efficiency of normal serum in blocking mouse reagin-induced PCA in rats. Balb/c and C3H/A strains showed higher susceptibility to passive sensitization and the efficiency of normal serum in blocking rat PCA lower than strain 129. The serum factor responsible for rat PCA inhibiting effect was heat-labile. The anaphylactic response of peritoneal mast cells of inbred strains to the challenge with anti-mouse IgE and anti-rat IgE in vitro was low in Balb/c mice, as compared with the response of C57BL/6J and 129 mice. The results suggest, that non-specific IgE present in the serum or bound to the mast cells may be one of the factors determining the susceptibility of mouse strains to reagin-induced passive sensitization. However, the results obtained with C57BL/6J strain suggest the existence of other factors effecting this susceptibility.     

Induction of humoral immunity and pulmonary mast cells in mice and rats after immunization with aerosolized antigen.

Rats (BN X Wistar) and mice (CBA/Ca) were immunized by exposure in 10-day periods to an aerosol of ovalbumin (OA). In rats this immunization resulted in IgE antibodies detectable at very low levels in bronchial washings, whereas IgG, IgA and IgM antibodies were recorded both in serum and in bronchial washings. In mice, exposure to aerosolized antigen resulted in specific IgE and IgG antibodies in serum. The levels of IgM antibodies were low and no IgA antibodies could be recorded with the enzyme-linked immunosorbent assay (ELISA). Histological examination of lung tissue from immunized rats and mice revealed increased numbers of cells with characteristics of both immature and mature mast cells. In addition, in the rats these cells were more closely located to the bronchi in immunized than in control animals. In the latter animals the mast cells were located around the blood vessels. Immature mast cells were located in the bronchiole-associated lymphatic tissue (BALT) which showed a marked proliferation in immunized animals. The findings indicate that sensitization via the airways provides possibilities to develop a model in rodents for studies of IgE-mediated allergy in the lung.     

Reduced tracheal mast cell numbers in adjuvant-sensitized rats associated with reduced pulmonary reactivity.

PVG/c inbred rats were sensitized by intra-peritoneal injection of DNP 19-ovalbumin with or without heat-killed Bordetella pertussis as adjuvant. The use of adjuvant was associated with a greater serum IgE, greater cutaneous immediate hypersensitivity, but no increase in immediate pulmonary reactivity to aerosol challenge. The number of mast cells in the trachea of adjuvant-sensitized rats was significantly reduced when compared with both unsensitized and allergen sensitized rats but this reduction in mast cell number did not occur in the skin. The reduction in tracheal and possibly other airway mast cells may explain the failure of the adjuvant sensitized rats to show increased pulmonary reactivity despite the increased serum IgE. The B. pertussis may have acted on the mediastinal lymph nodes, which drain both the peritoneal cavity and the lungs, to produce the observed reduction in tracheal mast cells.