Pascal Poncet

Immunologic Stimulation of Mast Cells Leads to the Reversible Exposure of Phosphatidylserine in the Absence of Apoptosis

Background: Loss of phospholipid asymmetry represents one of the hallmarks of apoptosis and results in the surface exposure of phosphatidylserine (PS) which can be indirectly monitored by the calcium-dependent binding of annexin V. Methods and Results: Here, we provide evidence that the IgE-dependent stimulation of a rat mast cell line, as well as murine and human nontransformed mast cells, leads to the exposure of PS at the plasma membrane. The appearance of PS was quantitatively related to allergic mediator release. Pharmacological agents that prevent stimulus-secretion coupling blocked PS cell surface exposure and calcium ionophore-induced PS appearance, suggesting that it is a direct consequence of exocytosis rather than early signaling events initiated by the aggregation of the high-affinity IgE receptor (FcεRI). The surface exposure of PS in mast cells was reversible even in the continuous presence of stimulus and was not associated with the appearance of apoptotic nuclei, demonstrating that it was independent of physiological cell death. Conclusions: In addition to providing a means of monitoring exocytosis at the single cell level, our results indicate that PS externalization in mast cells is not necessarily related to apoptosis but could be an important feature of the degranulation process.

MHC class II-dependent activation of CD4+ T cell hybridomas by human mast cells through superantigen presentation.

Human mast cells (MC) were examined for expression of MHC class II antigens and for their ability to activate CD4+ T cell hybridomas through presentation of superantigen (SAg). HMC‐1, a leukemic immature MC line expressing class II Ags, was shown to efficiently present the staphylococcal enterotoxin B (SEB) SAg to responding T cell hybridoma on treatment with interferon‐γ (IFN‐γ), which up‐regulated class II molecules. The study was then extended to human normal MC. Almost pure (>99%) cord blood‐derived MC (CBMC) were shown to express class II Ags (HLA‐Dr and HLA‐DQ) and CD80, which were up‐regulated by IFN‐γ treatment and, to a lesser extent, by interleukin‐4 (IL‐4) and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). CBMC directly activated CD4+ T cell hybridomas through presentation of SEB and TSST1 SAgs. The production of IL‐2 required a cell‐to‐cell contact between T cells and CBMC and it was inhibited by anti‐class II antibodies. Furthermore, an additional pretreatment of CBMC by IFN‐γ or GM‐CSF or IL‐4 had no effect on their presenting efficiency. This previously unknown function of human MC, i.e., MHC class II‐dependent activation of CD4+ T cells, may be critical in subsequent cellular activation events because colocalization of mast and T cells is frequently observed at sites of antigen entry. J. Leukoc. Biol. 66: 105–112; 1999.

Circulating human basophils lack the features of professional antigen presenting cells

Recent reports in mice demonstrate that basophils function as antigen presenting cells (APC). They express MHC class II and co-stimulatory molecules CD80 and CD86, capture and present soluble antigens or IgE-antigen complexes and polarize Th2 responses. Therefore, we explored whether human circulating basophils possess the features of professional APC. We found that unlike dendritic cells (DC) and monocytes, steady-state circulating human basophils did not express HLA-DR and co-stimulatory molecules CD80 and CD86. Basophils remained negative for these molecules following stimulation with soluble Asp f 1, one of the allergens of Aspergillus fumigatus; Bet v 1, the major birch allergen; TLR2-ligand or even upon IgE cross-linking. Unlike DC, Asp f 1-pulsed basophils did not promote Th2 responses as analyzed by the secretion of IL-4 in the basophil-CD4+ T cell co-culture. Together, these results demonstrate the inability of circulating human basophils to function as professional APC.

Monoclonal autoantibodies against mouse red blood cells: a family of structurally restricted molecules.

Cultured mouse peritoneal cells from unstimulated mice developed plaque-forming activity against isologous bromelain-treated erythrocytes. Several IgM monoclonal autoantibodies obtained by fusion of peritoneal cells from NZB or CBA origin with BALB/c myeloma cells were purified by affinity chromatography on trimethyl ammonium (TMA) column on the basis of their cross-reactivity with TMA, phosphorylcholine (PC) or choline haptens. Binding affinity for PC hapten was of the order of 10(3) M-1. Idiotypic studies with a polyclonal rabbit anti-idiotypic reagent revealed strong cross-reactions with all hybridoma autoantibodies thus far tested. In addition, the rabbit anti-idiotypic serum detected idiotypes or cross-reactive idiotypes in the sera of NZB and CBA as well as BALB/c mice. N-terminal amino acid sequence analyses of three hybridoma autoantibodies from NZB mice and one from CBA mice were carried out. The sequences of the first 32 residues of the four heavy chains showed that three were identical while one had one amino acid interchange; they belong to the VHIII-subgroup. The light chains were identical in the first 35 residues with the exception of a substitution at position 3 in two light chains and are members of the VK-9-subgroup. These results entirely support the idiotypic data. These monoclonal autoantibodies from NZB and CBA mice although isolated and eluted from PC-related haptens do not have any apparent structural nor idiotypic relationship to PC-specific antibodies. Idiotypic and V-region N-terminal sequence data suggest that these autoantibodies constitute a highly restricted family of molecules likely to be encoded by unique germ-line genes which may be expressed as such or as somatic variants in different mouse strains.

Allergomic study of cypress pollen via combinatorial peptide ligand libraries

Although Cupressus sempervirens (Cups) pollen represents one of the main aeroallergens in southern Europe, only two Cups allergens have yet been identified and reported: Cup s 1 and Cup s 3. The aim of this study was to identify allergens in cypress pollen using an immuno-proteomic approach. A sequential pollen protein extraction was developed and supplemented by a combinatorial peptide ligand library (CPLL) treatment to select low-abundance species. Control extracts and CPLL eluates have then been resolved by 1-DE and 2-DE gel electrophoresis, blotted and confronted with sera from cypress allergic patients. Extracted proteins including IgE-binding components were identified using nanoLC-MS/MS analysis. A total of 108 unique gene products were identified analyzing the eluates and control loaded onto 1-DE SDS-PAGE. Forty proteins were identified in control samples and 68 supplementary species upon CPLL treatment. Out of the 12 IgE-binding proteins characterized in 2-DE gels, 9 were already reported as allergens in various sources including the two major known allergens of Cupressaceae (groups 1 and 2). Three IgE-binding proteins, not previously reported as allergens, are newly described. The improvement in protein extraction combined with the enrichment of low-abundance species allowed us to extend the repertoire of potential cypress pollen allergens.

Anti-histone autoantibodies react specifically with the B cell surface

In an attempt to induce an immune response against Mls-1a antigens by immunizing C57B1/6 mouse (Mls-1b) with purified B cells from DBA/2 mouse (Mls-1a), we generated a panel of monoclonal antibodies from which the 5B9.6 mAb, taken as a representative antibody, was thoroughly investigated. This antibody specifically reacts with B cells from all mouse strains studied including C57Bl/6 mice as shown by FACS analysis of double-antibody labelled spleen cells. Using enzyme immunoassays and immunoprecipitation techniques, 5B9.6 mAb was found to be specific for histones. Amino acid sequence analysis of a peptide derived from a 5B9.6-immunoprecipitated polypeptide from DBA/2 B cells showed a 100% homology with a sequence within H2B histones. Furthermore, 5B9.6 mAb was able to interact with the cell surface of 7OZ/3 cell line, known as a typical pre-B cell line. The presence of histones can be modulated on the surface of 7OZ/3 cells since this antigen was upregulated after exposure of these cells to a cocktail of IL-1 and cAMP. Finally, 5B9.6 mAb was shown to interact with freshly isolated B cells from human peripheral blood.

A Review of the Effects of Major Atmospheric Pollutants on Pollen Grains, Pollen Content, and Allergenicity.

This review summarizes the available data related to the effects of air pollution on pollen grains from different plant species. Several studies carried out either on in situ harvested pollen or on pollen exposed in different places more or less polluted are presented and discussed. The different experimental procedures used to monitor the impact of pollution on pollen grains and on various produced external or internal subparticles are listed. Physicochemical and biological effects of artificial pollution (gaseous and particulate) on pollen from different plants, in different laboratory conditions, are considered. The effects of polluted pollen grains, subparticles, and derived aeroallergens in animal models, in in vitro cell culture, on healthy human and allergic patients are described. Combined effects of atmospheric pollutants and pollen grains-derived biological material on allergic population are specifically discussed. Within the notion of “polluen,” some methodological biases are underlined and research tracks in this field are proposed.

Polygalacturonase (pectinase), a new oilseed rape allergen

Background:  Type I hypersensitivity to rapeseed pollen allergens was described as the result of a cross‐sensitization with various pollens that could constitute an aggravating factor in birch or grass pollen allergies. Recently, a few rapeseed pollen allergens were described. The aim of the present work was to identify new rapeseed pollen allergens by using two‐dimensional gel analysis, microsequencing, and mass spectrometry.

Proteomics of cypress pollen allergens using double and triple one‐dimensional electrophoresis

Italian cypress (Cupressus sempervirens, Cups) pollen causes allergic diseases in inhabitants of many of the cities surrounding the Mediterranean basin. However, allergens of Cups pollen are still poorly known. We introduce here a novel proteomic approach based on double one‐dimensional gel electrophoresis (D1‐DE) as an alternative to the 2‐DE immunoblot, for the specific IgE screening of allergenic proteins from pollen extracts. The sequential one‐dimensional combination of IEF and SDS‐PAGE associated with IgE immunoblotting allows a versatile multiplexed immunochemical analysis of selected groups of allergens by converting a single protein spot into an extended protein band. Moreover, the method appears to be valuable for MS/MS identification, without protein purification, of a new Cups pollen allergen at 43 kDa. D1‐DE immunoblotting revealed that the prevalence of IgE sensitization to this allergen belonging to the polygalacturonase (PG) family was 70% in tested French allergic patients. In subsequent triple one‐dimensional gel electrophoresis, the Cups pollen PG was shown to promote lectin‐based protein‐protein interactions. Therefore, D1‐DE could be used in routine work as a convenient alternative to 2‐DE immunoblotting for the simultaneous screening of allergenic components under identical experimental conditions, thereby saving considerable amounts of sera and allergen extracts.

IgE Reactivity to Common Cypress (C. Sempervirens) Pollen Extracts: Evidence for Novel Allergens

BackgroundCypress pollen is becoming an increasing cause of respiratory allergy in some regions worldwide.ObjectiveThe aim of this study was to determine some of the main allergens implicated in the common cypress (C. sempervirens) pollen allergy.MethodsPollen extracts were optimized by using some detergents and chaotropes in order to solubilize both water and non-water soluble proteins. C. sempervirens pollen extracts were resolved by one and two dimensional electrophoresis and assayed with sera of allergic subjects.ResultsFive predominant allergens with apparent molecular masses ranging from 14 to 94 kDa were detected. Two principal IgE-binding patterns were clearly distinguishable: a first one represents patients with a heterogeneous IgE reactivity to several allergens (pI 3.5-8.5) with molecular masses ranging from 35 to 94 kDa (HMW). The second one corresponds to little less than 50 percent of tested patients with specific IgE binding to 2-3 spots (pI 10-11) of about 14 kDa and weak or no reactivity to HMW allergens.ConclusionThe extraction of water insoluble proteins allows the revelation of novel allergens as well as different allergen sensitization patterns in the C. sempervirens pollen allergy. These novel IgE reactive components may subsequently be applied to expand the panel of well-defined cypress pollen molecules for a more efficient allergen-based diagnosis and therapy.