B. David

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.

Capacity of mouse mast cells to prime T cells and to induce specific antibody responses in vivo

Mouse, human and rat mast cells have been shown to express major histocompatibility complex II molecules and present antigens to specific T‐cell hybridomas in vitro. The purpose of our investigation was to determine whether mouse mast cells are able to initiate specific immune responses in vivo. Induction of anti‐dinitrophenyl (DNP) immunoglobulin G1 (IgG1) and IgG2a antibodies was performed by transferring ovalbumin (OVA)–DNP‐pulsed bone marrow‐derived mast cells (BMMC), B cells, or macrophages into naive mice which were boosted later with soluble antigen. Cultured spleen cells from immunized mice were tested for their cytokine content. Our data show that mast cells were by far better inducers of anti‐DNP IgG1 antibodies than were B cells and macrophages. In contrast, anti‐DNP IgG2a response induced by macrophages was much stronger than that obtained with mast cells whereas B cells were completely unable to elicit this response. In addition to a high index of cell proliferation, spleen cells from mast cell‐injected mice produced more interferon‐γ than those mice who received macrophages or B cells by two‐ to fivefold, and almost 10‐fold, respectively. Mast cell‐deficient Wf/Wf mice were compared with their normal +/+ littermates and with mast cell‐reconstituted Wf/Wf mice to develop delayed‐type hypersensitivity (DTH) reactions as well as humoral immune responses. Mast cell sufficient mice as well as mast cell‐reconstituted Wf/Wf mice developed significantly increased DTH reactions (P = 0·02, and 0·03, repectively) and higher anti‐OVA‐specific antibody responses as compared with Wf/Wf mice. Our data suggest that mast cells have the potential to up‐regulate both humoral and cellular immune responses in vivo.

FcεRI-mediated antigen endocytosis turns interferon-γ-treated mouse mast cells from inefficient into potent antigen-presenting cells

Previous studies in our laboratory have shown that bone‐marrow‐derived mast cells (BMMC) could present immunogenic peptides, from soluble antigens endocytosed through fluid phase, only if they were subjected to a 48‐hr treatment with interleukin‐4 (IL‐4) and granulocyte–macrophage colony‐stimulating factor (GM‐CSF). In contrast to GM‐CSF, interferon‐γ (IFN‐γ) which highly upregulates major histocompatibility complex (MHC) class II expression, completely inhibits the generation of immunogenic peptides. We have used this model to study the role of FcεRI‐mediated antigen internalization in the regulation of the antigen‐presenting function of IFN‐γ‐treated mast cells. Here, we report that FcεRI can reverse the IFN‐γ‐treated mast cells from inefficient to highly efficient antigen‐presenting cells. Inhibition of the antigen presenting capacity by piceatannol, a protein tyrosine kinase (PTK) syk inhibitor, indicates that this is an active process resulting from immunoglobulin E (IgE)–antigen–FcεRI engagement which involves tyrosines found in the immunoreceptor tyrosine‐based activation motif (ITAM) embedded in the cytoplasmic tail of the FcεRI β and γ chains. Antigen‐presenting function was also shown to require the activation of phosphatidyl inositol 3 (PI3) kinase, downstream of PTK syk phosphorylation, since this activity was completely blocked by wortmannin, a PI3 kinase inhibitor. These data suggest that signalling generated by FcεRI provides mast cells with IgE‐mediated enhanced antigen presentation to T cells and emphasize a so far unknown immunoregulatory mast‐cell function that might take place in inflammatory sites.

Comparison of Conidial and Mycelial Allergens of Alternaria alternata

Alternaria allergens, separated by SDS polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane, were identified using sera from Alternaria-allergic patients. 30 Alternaria components of spore and mycelium, ranging in molecular weight from more than 150 to 12 kD, bound IgE antibodies. 15 were detected by more than 25% of the sera and only 4 (85, 56, 42, 31 kD) by more than 50% of the sera. Among these four major allergens, the 56-kD component was present mostly in the spore extract, the 85- and 42-kD fractions in the mycelium extract and the 31-kD in both extracts. The 31-kD component showed the highest IgE binding and the highest frequency of binding (95% with mycelium extract and 79% with spore extract). It was composed of two subunits.

Lymphocyte proliferative responses to the purified Dermatophagoides farinae major allergen in untreated and hyposensitized atopic patients

We investigated the proliferative response of lymphocytes from mite‐sensitive patients (RAST D.far > 3.5 PRU/ml) in the presence of the major allergen Der.f.I purified from Dermatophagoides farinae. Comparative studies were carried out with peripheral blood mononuclear cells from non‐atopic donors (RAST = 0), and from patients undergoing hyposensitization treatment (5 to 24 months). According to Students t‐test, there was no significant difference in the Der.f. I‐induced proliferation of peripheral blood mononuclear cells from normal donors, untreated atopic patients and hyposensitized patients. In conclusion, it was impossible to discriminate between normal donors, atopic patients and hyposensitized patients with regard to their circulating lymphocyte responses to the purified major allergen Der.f.I.

Comparison of the allergenic potency of spores and mycelium of Cladosporium.

The allergenic potency of spore and mycelium extracts of Cladosporium was estimated by RAST, RAST inhibition and PCA tests. Spores contained a concentration of allergens higher than mycelia. Results of PCA tests suggested that spores contained specific allergens. However, in a comparative study of extracts from different species of Cladosporium animal and human models gave different estimates of the allergenic potency of the different species. In spite of these variations it was shown that extracts from spores of Cladosporium contained the highest amount of Cladosporium allergens.