IgE receptor type I-dependent regulation of a Rab3D-associated kinase: a possible link in the calcium-dependent assembly of SNARE complexes.

Following activation through high affinity IgE receptors (FcepsilonRI), mast cells release, within a few minutes, their granule content of inflammatory and allergic mediators. FcepsilonRI-induced degranulation is a SNARE (soluble N-ethylmaleimide attachment protein receptors)-dependent fusion process. It is regulated by Rab3D, a subfamily member of Rab GTPases. Evidence exists showing that Rab3 action is calcium-regulated although the molecular mechanisms remain unclear. To obtain an understanding of Rab3D function we have searched for Rab3D-associated effectors that respond to allergic triggering through FcepsilonRI. Using the RBL-2H3 mast cell line we detected a Ser/Thr kinase activity, termed here Rak3D (from Rab3D-associated kinase), because it was specifically co-immunoprecipitated with anti-Rab3D antibody. Rak3D activity, as measured by its auto- or transphosphorylation, was maximal in resting cells and decreased upon stimulation. The down-regulation of the observed activity was blocked with EGTA, but not with other degranulation inhibitors, suggesting that its activity functions downstream of calcium influx. We found that Rak3D phosphorylates the NH(2)-terminal regulatory domain of the t-SNARE syntaxin 4, but not syntaxin 2 or 3. The phosphorylation of syntaxin 4 decreased its binding to its partner SNAP23. Thus, we propose a novel phosphorylation-dependent mechanism by which Rab3D controls SNARE assembly in a calcium-dependent manner.

Soluble NSF attachment protein receptors (SNAREs) in RBL-2H3 mast cells: functional role of syntaxin 4 in exocytosis and identification of a vesicle-associated membrane protein 8-containing secretory compartment.

Mast cells upon stimulation through high affinity IgE receptors massively release inflammatory mediators by the fusion of specialized secretory granules (related to lysosomes) with the plasma membrane. Using the RBL-2H3 rat mast cell line, we investigated whether granule secretion involves components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery. Several isoforms of each family of SNARE proteins were expressed. Among those, synaptosome-associated protein of 23 kDa (SNAP23) was central in SNARE complex formation. Within the syntaxin family, syntaxin 4 interacted with SNAP23 and all vesicle-associated membrane proteins (VAMPs) examined, except tetanus neurotoxin insensitive VAMP (TI-VAMP). Overexpression of syntaxin 4, but not of syntaxin 2 nor syntaxin 3, caused inhibition of FcepsilonRI-dependent exocytosis. Four VAMP proteins, i.e., VAMP2, cellubrevin, TI-VAMP, and VAMP8, were present on intracellular membrane structures, with VAMP8 residing mainly on mediator-containing secretory granules. We suggest that syntaxin 4, SNAP23, and VAMP8 may be involved in regulation of mast cell exocytosis. Furthermore, these results are the first demonstration that the nonneuronal VAMP8 isoform, originally localized on early endosomes, is present in a regulated secretory compartment.

Mapping of Dermatophagoides farinae mite allergens by two-dimensional immunoblotting.

BACKGROUND: Allergens from the house dust mite Dermatophagoides farinae are responsible for frequent respiratory allergic disorders, but only 3 groups of these allergens are well characterized. OBJECTIVE: This study was performed to complete the repertoire of D farinae allergens using two-dimensional (2-D) electrophoresis. METHODS: D farinae mite allergens, extracted from whole cultures in the presence of a mild detergent, were separated by 2-D electrophoresis with subsequent immunoblotting. IgE-binding proteins were detected with individual mite-sensitive patient sera and the anti-D pteronyssinus human serum pool. Allergens were identified by an inhibition immunoblot test, by means of specific mAbs, or by biochemical characterization. The internal peptides of 2 allergens were microsequenced. RESULTS: 2-D immunoblotting with individual patient sera showed a marked heterogeneity in the isoelectric point of the allergens, as well as differences in the individual IgE-binding patterns. In addition to identification of allergens Der f 1, Der f 2, and Der f 3, new allergens have been characterized as Der f 4, Der f 5, and 2 high molecular mass allergens. Microsequencing of peptides from the latter allergens revealed significant homologies with allergen Mag 3 from D farinae and with a chitinase from prawn Penaeus japonicus. CONCLUSION: 2-D electrophoresis with subsequent immunoblotting and protein microsequencing allowed characterization of a more complete repertoire of D farinae allergens and their multiple isoforms, and identification of six new allergens.

Recombinant murine (rm) interleukin (IL)-5 enhances the eosinophil peroxidase content in cells cultured in vitro compared with rmIL-3 and granulocyte-macrophage colony-stimulating factor.

We previously reported an in vitro bioassay permissive to eosinophil production from naive mouse bone marrow cells. Based on cytosmear analysis, recombinant murine (rm) interleukin (IL)-3 combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) provided better quantitative cellular yields compared with rmIL-5. In the present study, we compared the eosinophil peroxidase (EPO) content obtained in both in vitro conditions using microtitration. Parallel with the EPO assay, the percentage of eosinophils present in cultures was determined by cytosmear analysis. Cells incubated with IL-5 displayed significantly higher EPO activity than those cultured in the presence of IL-3 and GM-CSF. At the end of the culture period, cells were further enriched in eosinophils as determined by centrifugation gradient. Enhanced amounts of cellular EPO were detected when cultures were performed in the presence of IL-5. With regard to the correlation between eosinophil maturation and EPO acquisition, eosinophil cells cultured with rmIL-5 expressed significantly more EPO activity than those generated in the presence of rmIL-3 and GM-CSF.

Involvement of the ras-like GTPase rab3d in RBL-2H3 mast cell exocytosis following stimulation via high affinity IgE receptors (Fc epsilonRI).

Aggregation of high affinity IgE receptors (Fc epsilonRI) expressed on mast cells and basophils is a potent stimulus for the release of inflammatory mediators from cytoplasmic granules. Fc epsilonRI-dependent exocytosis requires activation of protein kinase C and mobilization of calcium from intra- and extracellular stores. However, how these events ultimately regulate the membrane fusion step between cytoplasmic granules and the plasma membrane still remains unclear. In this study, we investigated the role of the small GTPases of the rab3 subfamily in the regulated exocytosis following stimulation of rat basophilic leukemia cells (RBL-2H3). Analysis using reverse-transcriptase-based PCR showed that RBL-2H3 cells expressed rab3a and rab3d isoforms, with a predominance of rab3d at the mRNA level. Investigation of the subcellular distribution using isoform-specific Abs demonstrated that the majority of rab3a was expressed in the cytosol, whereas rab3d was found predominantly in the membrane fraction. To determine whether these proteins play a role in Fc epsilonRI-triggered exocytosis, we established RBL-2H3 transfectants that overexpressed wild-type and expressed GTP-binding mutant forms (N135I) of rab3a and rab3d. Whereas expression of rab3a proteins did not significantly affect degranulation as tested by beta-hexosaminidase release, those of both wild-type and mutant rab3d proteins inhibited degranulation. Calculations of the initial fast and of the second slow release rates showed that they are both inhibited about twofold, suggesting that rab3d interferes with a rate-limiting step in Fc epsilonRI-stimulated exocytosis.

Human IgG monoclonal antibodies that modulate the binding of specific IgE to birch pollen Bet v 1.

Birch pollen allergy is a very frequent pathology in Europe and North America. More than 95% of the tree pollen allergic patients display IgE reactivity against Bet v 1, the major birch pollen allergen. Starting with PBL from a patient desensitized by immunotherapy, we have generated five B cell lines (BAB1 to BAB5) that secrete human IgG mAbs of high affinity for Bet v 1. Although competition studies indicated that these human IgG mAb recognized different epitopes, broad cross-reactivity was found with Bet v 1 homologous allergens present in tree pollens and plant-derived foods. When tested for interference with allergic patients' IgE, BAB1 inhibited (by 80-100%) the binding of IgE to nitrocellulose-blotted Bet v 1, while BAB2 enhanced it. The biologic significance of the ability of BAB1 to interfere with patients' IgE binding is indicated by the finding that BAB1 completely inhibited Bet v 1-induced histamine release from allergic patients' basophils. Allergen-specific human IgG mAbs such as BAB1, which presents high blocking activity in both immunochemical and cellular IgE competition experiments, might have therapeutical application.

Human auto-anti-idiotypic antibodies to mite-specific IgE can degranulate human basophils in vitro.

BACKGROUND: Anti-idiotypic antibodies (anti-Ids) to specific IgE antibodies are formed spontaneously during an anti-allergen immune response and can be induced by immunotherapy. Although anti-Ids can down-regulate the production of IgE antibodies, at least in experimental models, their possible role in the modulation of target cell reactivity remains ill-defined. OBJECTIVE: The capacity of human anti-Ids to modulate the release of histamine was examined in an in vitro system of human basophil degranulation. Anti-Ids were prepared from the serum of six Dermatophagoides pteronyssinus (Dp)-hypersensitive patients suffering from atopic dermatitis and who had never been desensitized. Basophils were obtained from the blood of atopic donors. The extent of histamine release was determined using a fluorometric assay. RESULTS: We show that: anti-Ids trigger the release of histamine in an allergen-specific, dose- and IgE-dependent manner; the release is not due to the presence of allergen and/or anti-IgE antibodies; and that the degranulating activity can be removed by absorption with affinity-purified anti-Dp antibodies of the corresponding patient. CONCLUSION: These results indicate that spontaneously produced human anti-Ids can modulate the reactivity of human basophils.

[Importance of measuring exposure to the principal indoor allergens in allergic asthma]. / Intérêt de la mesure de l'exposition aux principaux allergènes de l'environnement intérieur dans l'asthme allergique.

Domestic allergens, such as mite and cat allergens, are a leading cause of allergic asthma. Allergen exposure is a risk factor for sensitization. Allergens also play a major role in the development of inflammation and non specific bronchial hyperreactivity as well as in the apparition and modulation of symptomatic asthma. The development of new means of detecting allergens (i.e. immunochemical assays including monoclonal antibodies, quantitative and semiquantitative guanine measurements for mite allergens) has made possible to identify allergens sources and reservoirs. The form in which domestic allergens become airborne is important. The group I and II allergens from mites, the major cockroach allergens are carried on large particles (mean size > 10 microns diameter); in contrast, the major cat allergens are airborne on small particles (40% < 5 microns). Guanine, a metabolic excretion product of mites, is used as a marker for mite feces and is correlated with the presence of major allergens from mites. A colorimetric method (Acarex-TestR) provides a simple and inexpensive method of assaying indoor mite allergen exposure for both doctor and patient alike. By using Acarex-TestR it is possible to evaluate the mite allergen exposure for a population in a particular country. The detection of allergens sources and reservoirs, the quantification of domestic allergens has enabled the evaluation of the effect of a reduction in allergen exposure to be better assessed. Recognition of the risks, environmental control and reduction in allergen loads, should be among the objectives of asthma management.

Immunogenicity and antigenicity of synthetic peptides derived from the mite allergen Der p I.

As an immunogen must contain both B- and T-cell epitopes, small peptides are usually reported as non-immunogenic unless coupled to a protein carrier. In this study, the immunogenicity of the Der p I synthetic uncoupled peptides (p52-71, p89-104, p117-133 and p176-187) previously reported as B-cell epitopes, was evaluated. Different schedules of immunization were used. Results indicated that by using the Vaitukaikis' method three injections of the same peptide without protein carrier was sufficient to induce an specific anti-peptide IgG antibody response (evaluated by ELISA). Indeed, the 16-20 amino-acid long peptides p52-71, p117-133 and p89-104 were revealed highly immunogenic in rabbits. Furthermore anti-peptide p52-71 and p117-133 antibodies were shown by Western-blotting or by neutralization assay to recognize the Der p I molecule either in denaturated or native form as well as Der f I (major allergen of Dermatophagoides farinae). Finally, taking into account the location of Der p I-derived peptides in the three-dimensional model of Der p I, the antigenicity and immunogenicity of peptides were discussed.

Prediction of mite allergen levels by guanine measurements in house-dust samples.

We studied the sensitivity and specificity of guanine environmental tests in the evaluation of the two mite allergen levels, i.e. 2 micrograms/g and 10 micrograms/g of Der p I and Der f I, considered to be risk factors for sensitization or for the development of acute asthma. We examined 239 house-dust samples for Der p I and Der f I levels (ELISA) and guanine contents (semiquantitative guanine test and quantitative assays). All house-dust samples with class 2 or 3 guanine tests contained more than 2 micrograms/g of Der p I and Der f I. The probability that house-dust samples of class 2 contained more than 10 micrograms/g of mite allergens was 88%; it was 100% for house-dust samples of class 3. The probability that a house-dust sample of class 0 contained less than 2 micrograms/g of mite allergen was 87%. For each level of mite allergen, a ROC curve was constructed with the true positive rates and the false positive rates calculated by different cutoffs of guanine concentration. The cutoff point which gave the best compromise between sensitivity (76%) and specificity (89%) was 2100 micrograms/g for the threshold of 2 micrograms/g of Der p I and Der f I. For detection of a mite allergen > 10 micrograms, a guanine content of 3000 micrograms/g gave the best compromise between sensitivity (86%) and specificity (93%). In conclusion, the guanine test represents a satisfactory environmental test, inexpensive and simple, for predicting mite allergen levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of allergenic epitopes on Der f I, a major allergen of Dermatophagoides farinae, using monoclonal antibodies.

The antigenic and allergenic structure of Der f I, a major allergen of the house dust mite Dermatophagoides farinae (Df) was investigated by means of a panel of 11 selected monoclonal antibodies (mAb) obtained from BALB/c mice immunized with purified Der f I. The species specificity of these mAb, tested with Der f I and Der p I--the homologous allergen from Dermatophagoides pteronyssinus--was generally restricted to Der f I since 10 out of 11 mAb reacted only with this allergen. Epitope specificity of the mAb was determined by both competitive inhibition and sandwich ELISA experiments. The results indicated the presence of at least four non-overlapping, non-repeated antigenic sites on Der f I, which were recognized by one or several mAb (sites A, B, C and D). Comparative epitope specificity studies between human IgE antibodies and mice mAb were performed, on sera and basophils of Df sensitive patients, using different inhibition assays (ELISA and histamine release experiments). The degree of inhibition varied between the patients and upon the assay design. Most of the mAb tested were found to significantly inhibit the binding of human IgE to Der f I (p less than 0.01) when compared with Der p I specific mAb as a control. The mAb reacting with site A was found to be the most potent inhibitor, presenting a mean inhibition of up to 56% in ELISA as well as in histamine release experiments. The results show that both human IgE antibodies and mAb can be directed against identical or closely related epitopes of Der f I. Therefore anti-Der f I mAb constitute immunologic probes in further allergenic epitope and peptide analysis of this major mite allergen.

[Tool for the study of household allergens]. / Moyens d'étude des allergènes de l'environnement domestique.

Analysis of environmental allergens is a method used for research purposes and for the management of patients with allergic rhinitis or asthma. Until recently, only pollen counts and the determination of atmospheric fungal spores could be performed, but new techniques are now available which enable allergens that are present in the indoor environment to be identified and quantified. With the development of immunochemical techniques using antibodies directed specifically against the major allergens contained in house dust, numerous samples of house dust or environmental air can be analyzed. Alternatively, tests detecting markers of allergen-producing organisms (excretion and secretion products) can be envisaged for routine analysis of multiple house dust specimens. The new techniques should result in a better definition of tolerable allergen levels and of thresholds of sensitization or clinical manifestations. They also provide objective means of evaluating the effectiveness of measures aimed at removing allergens from household environments.

Guanine content and Dermatophagoides pteronyssinus allergens in house dust samples.

The study of the relationship between the guanine content and Dermatophagoides pteronyssinus (Dp) allergens in house dust samples is reported. Mattress and carpet dust of bedrooms from 22 different homes constituted the house dust samples. The guanine content was determined by quantitative measurements and the mite allergenicity by two immunochemical assays with a partially purified extract of Dp as internal reference: RAST inhibition and crossed and rocket line immunoelectrophoresis. A large scale range of guanine content was obtained among the 22 house dust samples studied (0.01 to 1.78 mg/0.1 gm of dust). Data of RAST inhibition, analyzed according to parallel line bioassay, demonstrated no significant difference between the slopes of the reference and the house dust sample lines, but a 100-fold variation in the relative Dp potencies was observed. By crossed and rocket line immunoelectrophoresis technique, the presence and the amounts of major Dp allergens (Der p I and Dp 4) were established in most house dust extracts. A significant correlation was found between the guanine content of the house dust samples and their relative Dp potencies (r = 0.86) on the one hand, and with their relative content of Der p I and Dp 4, two major Dp allergens (r = 0.75 and r = 0.74, respectively) on the other hand; in each case, a quantitative relationship was established. These results suggest that the guanine determination could assess mite allergens in house dust and may be a useful tool in large-scale investigations of house dust.

Guanine and mite allergenicity in house dust.

Guanine is the major nitrogenous waste product in arachnids. It may serve as an indicator for allergenic mite faecal pellets. The present study assesses the correlation between the mit allergenicity of different house dust (HD) samples and their guanine content. Guanine content in HD samples was evaluated either by the Acarex-Test or determined quantitatively, especially for in-vitro methods. Using intradermal tests in selectively sensitized Dermatophagoides pteronyssinus (Dpt) patients, we evaluated the mite allergenicity of eight extracts obtained from HD samples with varying guanine contents. The weal area appeared to be proportional to the HD samples' guanine content. A three-fold increase in skin reactivity was observed when the guanine content varied from 0.06% to more than 1%. Patients allergic to HD but not to Dpt had no significant reactions with HD extract prepared from a guanine-rich HD sample. For eighteen different HD samples coupled to paper discs, the percentage Dpt RAST binding values obtained with a Dpt-selectively sensitized patient serum pool was correlated with the guanine concentrations of HD samples (P less than 0.01). The potencies of different HD extracts obtained from HD samples that varied in guanine content were also clearly distinguished on histamine-release titration curves. House dust obtained from homes at high altitude or from hospitals was generally guanine free. The HD sample guanine contents in homes where twelve Dpt-sensitized patients experienced symptoms were significantly higher than in homes where these patients were symptom free. The guanine colour test, a quick and easy technique, represents an important new development in indoor environmental investigations. It makes it possible to demonstrate mite faecal exposure and can be used to monitor the effectiveness of mite allergen avoidance measures.

Relationship between specific circulating IgE, basophil cell-bound IgE and histamine release induced by purified allergens of Dermatophagoides farinae.

In this study we analysed the presence and proportions of IgE specific for Df 6 and Df 11, two purified allergens of Dermatophagoides farinae. The characterisation of serum IgE and cell-bound IgE for 3 D. farinae-sensitive patients were performed by CRIE, RAST and PRIST assays. Furthermore the basophils from these same patients were studied by histamine release assays in the presence of Df 6 and Df 11. All the individual patient's cell and serum IgE samples displayed the presence of IgE antibodies specific for Df 6 and Df 11, but the relative quantities of the IgE for these two specificities were characteristic of each patient. The ratios (Specific IgE for Df 11) : (Specific IgE for Df 6) (ratio 11 : 6) were similar in the serum and on the cells for an individual patient. As judged by histamine release assays, the basophil sensitivities towards Df 6 and Df 11 were very different from one atopic patient to another. Moreover cell sensitivities reflected the proportion of Df 6- and Df 11-specific IgE antibodies found in the serum and in the cell eluate.

Comparison of antigenic and allergenic composition of two partially purified extracts from Dermatophagoïdes farinae and Dermatophagoïdes pteronyssinus mite cultures.

Dermatophagoïdes farinae (Df 80d) and Dermatophagoïdes pteronyssinus (Dp 80d) extracts were analyzed for their antigenic and allergenic composition by means of crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE). By CIE, 11 antigens could be numbered in Df 80d (Df 1 to Df 11) and seven antigens in Dp 80d (Dp 1 to Dp 7). This technique allowed us also to define antigens with common as well as specific parts for the two mite species. Among the antigens of D. farinae and D. pteronyssinus, only the antigen corresponding to Df 5 and Dp 5 seems to bear common epitopes to the two mite species, whereas Df 6 and Dp 4 appear to bear, respectively, specific epitopes of each species. Moreover, Df 11 appears to bear specific epitopes of D. farinae, although it shows a partial identity with Dp 7. By CRIE, on 20 mite-sensitive patients' sera, we identified, for each mite extract, the allergens responsive to human specific IgE. The allergograms show that the majority of mite-sensitive patients react with Df 11 and Df 6 and with Dp 7 and Dp 4. Thus, these antigens can be considered as major allergens. The minor allergens were also identified. None of these antigens was recognized by the control sera. Moreover, we observed that for one antigen (antigen 5) there exist antigenic determinants common to the two species of mites toward the rabbit serum and specific allergenic determinants to the human IgE response. A significant correlation was found between the specific IgE binding in CRIE and in RAST (Spearman coefficient: "rs" = 0.61 p less than 0.01 for Df; "rs" = 0.78 p less than 0.01 for Dp).

Antigens and allergens in Dermatophagoïdes farinae mite. I. Immunochemical and physicochemical study of two allergenic fractions from a partially-purified Dermatophagoïdes farinae mite extract.

The fractionation of a partially-purified extract of Dermatophagoïdes farinae mite culture has been undertaken by gel filtration. Two fractions were isolated. One, P25, is a protein-rich fraction with mol. wt about 25,000. The other, GP8, is a polysaccharide-rich fraction with mol. wt around 8,000. By crossed immunoelectrophoresis, we detected eleven antigens in the partially-purified dialysed D. farinae extract (Df 80d) as well as in P25 fraction. One of them, ag 11, seems the most important allergen since in crossed-radio immunoelectrophoresis experiments it displays the faster radiostaining, implying that it binds the greatest part of the mite-specific IgE present in a pool of sera from mite-sensitive patients. By crossed-line immunoelectrophoresis, we demonstrated the absence of ag 11 in GP8, in which only ag 5 and ag 6 were identifiable. By radioalloergosorbent tests (RAST), it was found that P25- and GP8- coated paper discs can fix specific IgE induced in the majority of D. farinae sensitive patients. Defining a 'major allergen' as an allergen to which the majority of sensitive patients develop specific IgE, both P25 and GP8 do appear to contain at least one major allergen. By RAST inhibition method, using Df 80d as a solid phase, the allergenic activity of P25 appeared as slightly higher than that of Df 80d, whereas GP8 displayed a very weak inhibitor capacity. Thus, the allergic specificity of GP8 differs from that of Df 80d or P25.