Identification of transient glycosylation alterations of sialylated mucin oligosaccharides during infection by the rat intestinal parasite Nippostrongylus brasiliensis.

The sialylation of the oligosaccharides from small-intestinal mucins during a 13-day infectious cycle was studied in Sprague-Dawley rats with the parasite Nippostrongylus brasiliensis. Sialic acid analysis and release, permethylation and analysis by GC-MS of the sialylated oligosaccharides isolated from the 'insoluble' mucin complex revealed a relative decrease (4-7-fold) of N-glycolylneuraminic acid compared with N-acetylneuraminic acid just before parasite expulsion. Northern blots showed that this effect was due to the decreased expression of a hydroxylase converting CMP-N-acetylneuraminic acid into CMP-N-glycolylneuraminic acid. Analysis of other rat strains showed that this parasite infection also caused the same effect in these animals. Detailed analysis of infected Sprague-Dawley rats revealed four sialylated oligosaccharides not found in the uninfected animals. These new oligosaccharides were characterized in detail and all shown to contain the trisaccharide epitope NeuAc/NeuGcalpha2-3(GalNAcbeta1-4)Galbeta1 (where NeuGc is N-glycolyl neuraminic acid). This epitope is similar to the Sd(a)- and Cad-type blood-group antigens and suggests that the infection causes the induction of a GalNAcbeta1-4 glycosyltransferase. This model for an intestinal infection suggests that the glycosylation of intestinal mucins is a dynamic process being modulated by the expression of specific enzymes during an infection process.

Recruitment, distribution and phenotypes of mast cells in interstitial cystitis.

PURPOSE: Interstitial cystitis (IC) is a chronic disabling condition of unknown etiology. One of its major characteristics is an increase in mast cells (MC) showing signs of activation. It has been suggested that the proteinase content defines two MC types: MC(TC), containing chymase and tryptase, and MC(T), which contains tryptase but lacks chymase. Here, we investigated the MC distribution and the MC proteinase expression in IC together with the tissue expression of the major MC growth factors, stem cell factor (SCF) and interleukin-6 (IL-6). MATERIALS AND METHODS: MC were enumerated in bladder specimens from patients with classic IC, nonulcer IC and controls. MC were visualized in terms of metachromasia, reflecting glycosaminoglycan content, and immunohistochemically, visualizing tryptase, chymase and IL-6 as well as the surface markers CD117 and SCF. RESULTS: Classic IC displayed a 6 to 10-fold increase of MC identified by proteinase content while in nonulcer IC there were twice as many MC as in controls. In contrast to nonulcer IC and controls, classic IC displayed an abundance of epithelial MC. Fewer CD117+ than proteinase+ MC were detected in IC but not in controls. Classic IC coexpressed SCF and IL-6 in the epithelium and displayed numerous SCF and IL-6+ cells in the mucosa and detrusor muscle, many of which were MC. CONCLUSIONS: Redistribution of MC into the epithelium and a high bladder wall MC density distinguish classic IC from nonulcer IC. Our findings suggest an SCF/IL-6-driven MC response in IC. They also indicate a downregulation of the SCF receptor in IC.

Regulation of IgE-receptor expression, IgE occupancy and secretory capacity of mast cells.

Mast cells play an important role in initiating and modulating allergic and inflammatory reactions. Their responsiveness is determined by three important factors: the expression of IgE receptors on the cell surface, the IgE occupancy of these receptors, and the intrinsic secretory capacity of the cells. In this review, we will summarise some findings relevant to these three aspects of mast cell function, and discuss possible regulatory mechanisms. It appears that the genetic background as well as environmental factors influence all three of these components of the response. T cells appear to play an important role in regulating the IgE-receptor expression and also, independently, the intrinsic secretory capacity of mast cells via an unidentified route, possibly involving the secretory signal transduction chain directly. IgE itself appears to have an important role in the regulation of IgE-receptor expression, as indicated by the upregulation of receptors in vitro in the presence of IgE, and the absence of IgE-binding capacity of mast cells in IL-4 gene knockout mice, lacking IgE production. The IgE-receptors of mast cells are saturated to a high degree under different normal conditions, without an obvious relation to antigenic stimulation, also in athymic animals. We have suggested that this basal IgE content on mast cells may be the result of an antigen-independent production of IgE directed by the mast cells themselves and serving regulatory purposes, modifying the secretory response and preventing a massive possibly harmful degranulation.

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.

Surface and gene expression of immunoglobulin E receptors on mast cells and mast-cell numbers in interleukin-4-gene knockout mice.

We quantified immunoglobulin E (IgE) on peritoneal mast cells of interleukin-4 (IL-4)-gene knockout (-/-) mice and wild-type (+/+) controls using a cytofluorometric method, and examined the expression of IgE receptors, estimated by quantifying the total binding of IgE on the mast cells of IL-4 (-/-) mice. The mast cells of IL-4 (+/+) mice, identified and measured using microscope fluorometry, had a fluorescence intensity five to six times higher than that of non-mast cells, while the mast cells obtained from IL-4 (-/-) mice had fluorescence intensities within the range of those of non-mast cells. Two weeks after an infection with Nippostrongylus brasiliensis, the fluorescence intensity of the mast cells of IL-4 (+/+) mice increased to a level about twice as high as that before immunization. However, no significant increase after infection was observed in IL-4 (-/-) mice. Furthermore, the mast cells of IL-4 (-/-) mice did not bind IgE when incubated with IgE at concentrations that saturated IgE receptors on the mast cells of wild-type controls, thereby indicating that the expression of IgE receptors on mast cells was impaired in the IL-4-deficient mice. Using a reverse transcription-polymerase chain reaction, we found gene expression of all three subunits (alpha-, beta- and gamma-chains) of the IgE receptor in IL-4 (-/-) like that in IL-4 (+/+) mice. The results thus suggest that the binding of IgE may be essential to induce the translation of mRNA to IgE-receptor proteins. We also observed that there were about twice as many peritoneal mast cells in the IL-4 (-/-) mice as there were in the IL-4 (+/+) mice, in both immunized and non-immunized animals. This was unexpected in view of previous findings suggesting that IL-4, in concert with stem cell factor and IL-3, stimulates the proliferation and differentiation of mast cells in vitro.

A kinetic analysis of the expression of mast cell protease mRNA in the intestines of Nippostrongylus brasiliensis-infected rats.

To study the kinetics and the phenotype of the mast cells (MC) arising during infection with the nematode Nippostrongylus brasiliensis, monospecific cDNA probes for nine different MC proteases were used in a Northern blot analysis of RNA from the small intestine of infected rats. The expression was analyzed at four individual time points during infection, day 0 (before infection), and days 7, 12 and 16 post infection. A dramatic increase in mRNA for rat mast cell protease (RMCP)-2, the major mucosal MC protease in the rat, was observed, beginning around day 7 after infection and peaking around day 12. At day 16 the expression was already beginning to decline. An almost identical pattern of mRNA expression was detected for the RMCP-8 subfamily of rat MC proteases (RMCP-8, -9 and -10) and for two additional rat serine proteases, the chymases RMCP-3 and -4. No simultaneous increase in the proteases known to be expressed preferentially by mature connective tissue MC (RMCP-1, -6 and -7) was observed. This is consistent with our finding that the expansion of MC in the intestines of parasite-infected animals was limited, almost exclusively, to the mucosal MC population. However, a minor increase in RMCP-5 and MC carboxypeptidase A (CPA) mRNA was detected at day 12 after infection, suggesting a derivation of mucosal MC from an expanding RMCP-5- and CPA-positive population of MC precursors.

Metaplastic transformation of urinary bladder epithelium: effect on mast cell recruitment, distribution, and phenotype expression.

Mucosal mast cells (MCs) are normally found in the connective tissue stroma but are redistributed into the epithelium in conditions associated with immunoglobulin E responses, such as allergic inflammation and nematode infections, as well as in interstitial cystitis, a condition of unknown etiology. The potential role of epithelium-derived factors in this response prompted this inquiry into growth and differentiation signaling in normal tissue as well as in tissues from five different metaplastic conditions of the urothelium (cystitic cystica, cystitis glandularis, colonic metaplasia, squamous cell metaplasia, and nephrogenic metaplasia). Expression of the two major human MC growth factors, stem cell factor (or kit ligand) and interleukin 6, was detected using immunohistochemistry. In the case of interleukin 6, its mRNA expression was also detected using in situ reverse transcription-polymerase chain reaction. Among the different metaplastic lesions, nephrogenic metaplasia was the only one associated with an abundance of MCs, which were distributed within or in close relationship to the epithelium. Unlike in the other types of metaplasia, the epithelium strongly co-expressed interleukin 6 and stem cell factor. The MCs expressed the stem cell factor receptor CD117 and exhibited a variable tryptase immunoreactivity, but lacked chymase. They also displayed a relative deficiency of granular glycosaminoglycan, as indicated by a lack of metachromasia, and were sensitive to strong aldehyde fixation. The findings suggest that the MC response in nephrogenic metaplasia may be the result of local epithelial stem cell factor/interleukin 6 expression.

Vitamin A deficiency increases inflammatory responses.

The authors studied the influence of vitamin A deficiency on immediate and delayed type hypersensitivity as well as granulocyte-mediated inflammatory reactions in vitamin A depleted and control rats. The number of circulating leucocytes was 43% higher in the vitamin A deficient than in the control animals. The leucocytosis was a result of a general increase of white blood cells and was not due to an increase in one particular type. The ratio between CD4+ and CD8+ T cells was unchanged. The vitamin A deficient rats had a four times higher T-cell proliferative response and a two times higher interferon-gamma production in vitro than the control animals. In accordance, the DTH reaction was consistently higher in the vitamin A deficient rats. The granulocyte dependent inflammation, induced by olive oil injection, was also strongly enhanced in the vitamin A deficient rats compared with the controls. In addition, the spontaneous release of nitric oxide from the peritoneal phagocytes was five times higher in the vitamin A deficient animals. The number of peritoneal mast cells was about one and a half times higher in the vitamin A deficient than in the control animals. The density of IgE-receptors on the mast cells, the IgE receptor occupancy and the histamine release from the mast cells did not differ between the groups, however. The vitamin A deficient immunized rats displayed a consistently stronger immediate skin reaction after intracutaneous antigen injection than the immunized control rats, despite lower IgE antibody levels. The skin reaction after intracutaneous injection of histamine was also significantly greater in the deficient animals. Despite the stronger reaction to antigen and histamine, the passive cutaneous anaphylaxis reaction was lower in the vitamin A deficient rats. In conclusion the study shows that vitamin A deficiency aggravates the clinical manifestations of inflammatory reactions. Thus, vitamin A deficiency might lead to a higher risk of acquiring irreversible tissue damage and disabling destruction.

Immune responses to a Nippostrongylus brasiliensis infection in athymic and euthymic rats: surface expression of IgE receptors, IgE occupancy and secretory ability of mast cells.

The significance of T cells in an allergy-related immune response was investigated. Athymic, nude (Lewis rnu/rnu) rats lacking T lymphocytes and euthymic (Lewis+/+) rats were infected with the nematode Nippostrongylus brasiliensis. The density and occupancy of IgE receptors on peritoneal mast cells were quantified using a cytofluorometric method. The secretory ability of the mast cells as a function of anti-IgE challenge was evaluated in terms of histamine releasability in vitro. It was found that the IgE receptors were markedly up-regulated and the IgE occupancy increased on the mast cells of both athymic and euthymic rats during the parasite infection. However, at its peak, IgE occupancy was significantly higher in euthymic rats than in athymic ones. To our knowledge, this is the first proof of the possibility of a T-cell-independent IgE response in vivo. The IgE-mediated histamine releasability of mast cells was significantly enhanced 1 week after the infection in euthymic rats but not in athymic ones. These results thus indicate that the IgE immune response can occur in the absence of, but is augmented by, T cells. They also suggest that the concept of the IgE response should be widened to comprise not only increased IgE production but also an up-regulation of Ig receptors and an increase in IgE occupancy on mast cells, as well as an increase in the secretory ability of these cells. The latter is T cell dependent, although it is not directly related to the density of IgE-receptor complexes on the mast cells but is more likely due to a stimulation of the post-receptor signal transduction mechanism.

T-cell-independent and T-cell-dependent IgE responses to the nematode Nippostrongylus brasiliensis: comparison of serum IgE and mast-cell-bound IgE.

The IgE immune response was studied in female athymic, nude (Lewis rnu/rnu) and euthymic (Lewis +/+) rats infected with the nematode Nippostrongylus brasiliensis. During the course of the infection, serum IgE levels were followed using an enzyme-linked immunosorbent assay technique (ELISA), while the surface expression and occupancy of IgE receptors on peritoneal mast cells were quantified using flow cytometry after immunolabelling with anti-IgE. The results show that the up-regulation of IgE receptors, which takes place on the mast cells of both athymic and normal rats during the early phase of the immune response, is more pronounced and longer-lasting in normal rats than in athymic ones, thereby suggesting that T cells are necessary for a full response to the parasite infection. The increased IgE occupancy observed on the mast cells during the early phase of the parasite immune response was not reflected in the serum IgE levels, which remained low during the entire infection period in athymic rats. In euthymic rats, on the other hand, there was a pronounced increase in serum IgE, as well as an increase in IgE occupancy on the mast cells, all reaching a peak level after 2 weeks of infection. However, there was no significant correlation between the serum IgE concentration and IgE occupancy or the density of IgE receptors on the mast cells of the individual euthymic rats. This indicates that the quantification of IgE occupancy on the mast cells may be a better way of detecting low-level IgE responses than the measurement of serum IgE. These findings, which were obtained in female Lewis rats, when compared with our previous findings in male rats of the same strain, suggest that sex differences may exist in terms of the intensity and duration of the IgE immune response to the parasite infection.

Surface expression of IgE receptors, IgE occupancy and histamine releasability of mast cells: influence of genetic and T cell factors.

IgE receptor density and IgE occupancy were quantified on individual peritoneal mast cells using cytofluorometry. The secretory capacity of the mast cells was analysed in terms of histamine release as a function of anti-IgE challenge. Histamine releasability was found to be largely a function of the number of IgE molecules on the mast cells. The genetic background influenced IgE receptor expression but also, independently, histamine releasability. Both IgE receptor density and IgE occupancy on the mast cells appeared to be T cell independent under normal conditions. Up-regulated IgE receptor expression and an increased IgE occupancy on the mast cells could be obtained in athymic rats in response to a Nippostrongylus brasiliensis infection, although it was not as pronounced as in euthymic rats. The secretory response was diminished in athymic rats, suggesting that T cell factor(s) enhance secretory signal transduction in the mast cells in vivo.

Influence of genetic and environmental factors on surface expression and occupancy of IgE receptors and on histamine releasability of mast cells.

The relationship between the IgE load on mast cells and their secretory capacity when challenged with anti-IgE was studied in peritoneal cells obtained from rats of three different strains, Hooded Lister (HL), Wistar Kyoto (WKY) and Sprague-Dawley (SD). IgE was determined cytofluorometrically after labelling with FITC-conjugated anti-IgE before and after the saturation of the IgE receptors to provide a measure of the surface expression of IgE receptors (number of receptors available for bindings) as well as the IgE occupancy of the receptors (native IgE content). The secretory capacity of the mast cells was examined in vitro in terms of histamine release as a function of anti-IgE concentration. Mast cells obtained from HL and WKY rats were found to carry significantly higher levels of IgE receptors and IgE than the mast cells of SD rats bred and raised under the same conventional laboratory conditions. The mast cells of SD rats kept under barrier-maintained conditions carried significantly less IgE than the mast cells obtained from SD rats kept under conventional conditions, but their IgE receptor levels were similar. The IgE-mediated histamine-releasing capacity of the mast cells, evaluated in terms of maximum release or of the slopes of regression lines (histamine release versus anti-IgE concentration), was positively correlated to the levels of native IgE and IgE receptors in the three strains of rat combined. The mast cells obtained from WKY rats showed the highest secretory capacity in the three strains of rat examined, significantly higher than the mast cells of HL rats, even though the latter displayed similar levels of IgE and IgE receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteinase content of mast cells of nasal mucosa; effects of natural allergen exposure and of local corticosteroid treatment.

The distribution and density of metachromatic cells (MCC) and mast cells containing chymase plus tryptase (MCTC) or tryptase alone (MCT) were studied in the nasal mucosa by dye-binding methods and immunohistochemical analysis. Biopsies were obtained from 17 subjects with birch pollen allergy before and during the peak season and from nine healthy controls. Six patients were treated with an intranasal glucocorticosteroid before and during the season in an open study. Hay fever patients, even when asymptomatic, showed signs of mast cell system activation, exhibiting an increased number of mast cells in the nasal epithelium. Basophils, lacking immunohistochemically detectable tryptase, were not a major component of the mast cell response. MCT, most conspicuous in the epithelium, were found to be the most frequent mast-cell type in the nasal mucosa of allergic, but not of normal, subjects. Only 33% of the epithelial, but 90% of the stromal, immunopositive cells in the atopic mucosa before as well as during the season were MCC. Intraepithelial MCT thus displayed a low capacity to stain metachromatically, indicating a relative deficiency of the glycosaminoglycan (heparin) component of the granules. Intraepithelial mast cells also appeared to be markedly sensitive to steroid treatment and aldehyde fixation. The findings suggest that the lack of chymase, the characteristic feature of MCT, may reflect a functional activation of the mast cells, rather than a stable phenotypic differentiation related to anatomic site.

Cytofluorometric quantification of IgE and IgE receptors on individual mast cells.

Information about the IgE receptor and IgE content of mast cells under different conditions in vivo is essential for further understanding the functions of the mast cell-IgE system. A cytofluorometric method for measuring cell-bound IgE on individual mast cells was therefore explored using peritoneal mast cells of Sprague-Dawley rats infected with the nematode N. brasiliensis and rat basophilic leukaemia cells (RBL-1) as experimental models. We systematically studied the effects of variables such as fixation, incubation time, temperature and concentrations of antibody on the fluorescence emission of the mast cells. Optimum conditions were selected for the quantitative measurement of IgE at the single-cell level using direct labelling with FITC-conjugated goat anti-rat IgE(Fc). Polystyrene beads with a defined fluorophor content and a fluorescent uranyl glass were used to standardise the measurement procedure. A linear relationship between fluorescence intensity and IgE concentration was obtained by fluorescence measurements on RBL-1 cells incubated in rat IgE. In the case of rat peritoneal mast cells it was possible to saturate the IgE receptors by incubating the mast cells in rat IgE. By measuring the mast cells before and after IgE incubation, the relative content of IgE, the relative number of IgE receptors and the degree of receptor saturation could be estimated. In this way we measured the IgE content of peritoneal mast cells of Sprague-Dawley rats maintained under pathogen-free conditions. The distribution of IgE content in the mast-cell populations was extremely variable. Up to 30% of the mast cells in individual populations contained little or no IgE, but very few if any of the cells lacked IgE receptors. On average, 60-70% of the receptors available for binding were occupied by IgE in these normal rats.

IgE receptors, IgE content and secretory response of mast cells in athymic rats.

Athymic, nude (Lewis rnu/rnu) and normal (Lewis +/+) rats were used to study the T-cell dependence of the regulation of IgE receptors and IgE content on mast cells in vivo. We estimated the number of IgE receptors and the IgE content on peritoneal mast cells using a cytofluorometric technique. The secretory capacity of the mast cells was measured in terms of histamine release as a function of anti-IgE concentration by incubation with antibodies in vitro. Two groups of rats, aged 6 and 13 weeks, were examined. The mast cells of the rats belonging to the older age group (both normal and athymic) had a higher total protein (equivalent to dry mass or size) and mediator content (heparin, histamine and 5-hydroxytryptamine) in accordance with previous findings. Mast cells of the athymic rats of both age groups contained similar levels of IgE receptors and IgE and did not differ in these respects from those of the normal rats. Of the IgE receptors available for binding, about 80% were occupied by IgE in mast cells of normal and of athymic rats in both age groups. The anti-IgE-induced histamine release of the mast cells was, however, significantly lower in athymic rats compared to the normal controls. A change in housing from barrier-maintained to conventional conditions for 2 weeks enhanced the IgE-receptor and IgE content, as well as the releasability of histamine of the mast cells of both athymic and normal rats. The basal level of IgE occupancy of the receptors on mast cells was therefore not impaired in the athymic rats, as might be inferred from previous findings of a T-cell dependence of the IgE immune response. The results further indicate that T-lymphocyte-derived cytokines appear not to be required for either the expression of IgE receptors or for their ability to acquire IgE on mast cells, but such factors seem to enhance the release of histamine following the cross-linkage of the IgE receptor on mast cells in normal conditions.

Application of an immunocolloid gold technique for the ultrastructural demonstration of IgE-receptor complexes on rat mast cells.

In previous works using cytofluorometry, we demonstrated a broad range of IgE and IgE-receptor levels within individual mast cell populations with a 60 to 80% occupancy of the IgE receptors on mast cells by native IgE. This study was performed in order to confirm our previous findings using an independent method and to visualize the distribution of IgE-receptor complexes on mast cells at an ultrastructural level. For this purpose an indirect immunocolloidal gold-labelling technique has been applied. By counting the number of labelled gold particles, a relative measure of IgE-receptor surface expression and IgE occupancy of the receptors could be obtained. With respect to mast cell morphology and anti-IgE binding specificity criteria, 1% glutaraldehyde + 4% paraformaldehyde (1:1, vol/vol) was found to be the best of the seven fixatives applied in this study. This technique revealed numerous gold particles on the surface of mast cells from barrier-maintained rats (26 +/- 11 per mast cell section, mean +/- SD). Increased numbers of gold particles were counted if the mast cells were incubated with rat myeloma IgE (20 micrograms/ml) (46 +/- 33 per mast cell section, mean +/- SD). There were significantly increased numbers of gold particles on the mast cells of rats infected with N. brasiliensis (126 +/- 30 per mast cell section, mean +/- SD). This indicates that some of the IgE receptors (about 50% of the total number of IgE receptors in this case) on mast cells were occupied by native IgE and that parasite infection significantly increased the number of IgE molecules on the surface of the mast cells. These results correspond with the findings we have made using the cytofluorometric technique and confirm the large individual variations in the density of IgE receptors and IgE among the mast cells of a given cell population. Macrophages, lymphocytes and eosinophils, carrying the low-affinity IgE receptors (Fc epsilon RII), contained less than 5 (normal rats after incubation in rat IgE) or 10 (nematode-infected rats) gold particles per cell section. We also observed some non-granulated lymphocyte-like cells which bound a large number of gold particles after incubation with rat myeloma IgE (20 micrograms/ml), indicating that they contained IgE receptors Fc epsilon RI). They were interpreted as mast cell precursors which have previously been shown to exist in the peritoneal cavity.