IL-3-producing basophils are required to exacerbate airway hyperresponsiveness in a murine inflammatory model.

BACKGROUND: Basophils are commonly associated with allergic responses because of their ability to produce large amounts of pro-Th2 cytokines and histamine. However, the mechanisms through which bone marrow-resident basophils (BMRB) become fully competent cytokine and histamine producers in response to IgE crosslinking are poorly understood. Here, we sought to determine the role of IL-3 in promoting pro-Th2 basophils. METHODS: BMRB and basophils exposed to IL-3 in vitro and in vivo were evaluated for their production of Th2 cytokines and histamine in response to FcεRI crosslinking on both protein and gene expression levels. In vivo relevance of our findings was assessed in a model of ovalbumin-induced allergic asthma using IL-3-deficient and wild-type mice in a protocol of adoptive basophil transfer. RESULTS: We show that BMRB and basophils previously exposed to IL-3 differ in their ability to generate cytokines (IL-4, IL-6, IL-13, and GM-CSF) and histamine in response to FcεRI crosslinking, reflecting two stages of maturation. Exposure to IL-3 initiated an autocrine loop of endogenous IL-3 production that enhanced histamine and cytokine production upon FcεRI crosslinking. This increased responsiveness required calcium flux and was dependent on calcineurin and store-operated calcium channels. Our findings are of pathophysiological relevance, as assessed by the failure of IL-3-deficient mice to develop airway hyperreactivity, which could be restored by adoptive transfer of IL-3-derived basophils recovered from wild-type mice. CONCLUSION: IL-3-dependent basophils promote Th2 allergic AHR, which designates the IL-3/basophil axis as a promising therapeutic target for the treatment of basophil-dependent asthma.

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.

Human serum IgE-mediated mast cell degranulation shows poor correlation to allergen-specific IgE content.

BACKGROUND: Although allergen-specific IgE content in serum can be determined immunochemically, little is known about the relationship between this parameter and the strength of the degranulation response upon allergen triggering. OBJECTIVES: Analyse the degranulation capacity of immunochemically defined purified and serum IgE after challenge with anti-IgE or allergen using a rat mast cell line (RBL) transfected with the alpha-chain of the human high-affinity IgE receptor (FcepsilonRI). METHODS: Purified IgE specific for 4-hydroxy-3nitrophenylacetyl, purified IgE of unknown specificity, and sera from allergic patients sensitive to Dermatophagoides pteronyssinus and Dactylis glomerata were assessed. Degranulation was measured by a beta-hexosaminidase release assay after anti-IgE or allergen-specific challenge. RESULTS: For purified monoclonal IgE a significant correlation (r = 0.97) was found between the proportion of bound allergen-specific IgE and the strength of the degranulation response. In contrast, no correlation (r = 0.27) was detected after sensitization with serum IgE. CONCLUSION: Our studies demonstrate that mast cell activation mediated through IgE from allergic patients is a result of complex relationships that are not only dependent on allergen-specific IgE content but also relate to the capacity to efficiently sensitize and trigger the signalling responses that lead to degranulation.

[Immunological and non immunological mechanisms in urticaria]. / Mécanismes immunologiques et non immunologiques des urticaires.

Urticaria involve mast cell activation which could be mediated by immunological or non-immunological mechanisms. Interaction of allergens with the IgE/IgE receptor at the surface of mast cells has been postulated as the main immunologic type of mast cell activation. However, recent experimental and clinical studies have highlighted the existence of other mechanisms involving specific antibodies and T cells. IgG antibodies of different specificities (anti-IgE and/or anti-IgE receptor autoantibodies) have been characterized in a subgroup of patients suffering from chronic "autoimmune" urticaria. Circulating immune complexes may activate mast cells by interaction with the membrane-bound receptor for IgG. Interaction of mast cells with specific T cells could induce mast cell activation. Thus, immune-mediated urticaria appears to be secondary to different types of mast cell activation which could explain the various clinical presentation of the disease.

Nonspecific B and T cell-stimulatory activity mediated by mast cells is associated with exosomes.

Bone marrow-derived mouse mast cells (BMMC) and mast cell lines P815 and MC9 have recently been shown to induce antigen-independent B and T lymphocyte activation. It has been demonstrated that a physical contact between mast cells and B and T lymphocytes is not necessary since mast cell supernatants contain full activity. Electron microscopy studies revealed the presence in mast cell supernatants of small vesicles called exosomes with a heterogeneous size from 60 to 100 nm of diameter. When cocultured with spleen cells, purified exosomes induce B and T cell blast formation, proliferation as well as IL-2 and IFN-gamma production. In contrast to P815 and MC9 mast cell lines, a pretreatment with IL-4 is required for BMMC to produce active exosomes. Structurally, these exosomes were found to harbor immunologically relevant molecules such as MHC class II, CD86, LFA-1 and ICAM-1. Here we provide for the first time the evidence that mast cells use exosomes as sophisticated messengers to communicate with cells of the immune system.

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-gamma 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, respectively) 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.

Mast cell-dependent B and T lymphocyte activation is mediated by the secretion of immunologically active exosomes.

Mitogenic activity of bone marrow-derived mouse mast cells and mast cell lines P815 and MC/9 on B and T lymphocytes is present in their culture supernatants. To identify this activity, mast cells were incubated in serum-free medium and the supernatant was subjected to differential centrifugation, which resulted in two fractions, the hypodense and dense fraction (pellet). When analyzed for their mitogenic activity on spleen cells, all activity was found to be associated with the dense fraction. Electron microscopy studies revealed the presence in this fraction of small vesicles called exosomes with a heterogeneous size from 60 to 100 nm of diameter. When cocultured with spleen cells, purified exosomes induced blast formation, proliferation, as well as IL-2 and IFN-gamma production, but no detectable IL-4. Similar data were obtained by injecting exosomes into naive mice. In contrast to mast cell lines, a pretreatment with IL-4 is required for bone marrow-derived mast cells to secrete active exosomes. Structurally, exosomes were found to harbor immunologically relevant molecules such as MHC class II, CD86, LFA-1, and ICAM-1. These findings indicate that mast cells can represent a critical component of the immunoregulatory network through secreted exosomes that display mitogenic activity on B and T lymphocytes both in vitro and in vivo.

In vitro and in vivo immunostimulatory potential of bone marrow-derived mast cells on B- and T-lymphocyte activation.

BACKGROUND: Mast cells, which play a unique role in inflammatory and allergic responses, have also been shown to actively participate to the build-up of protective host defense mechanisms. Recently, they have been shown to stimulate resting B cells and to form heterotypic aggregates with activated T cells, resulting in mast cell degranulation. OBJECTIVES: Our aim is to investigate the cytokine requirements and the mechanisms by which murine mast cells activate resting B and T lymphocytes. METHODS: Mouse bone marrow-derived mast cells (BMMCs) or peritoneal mast cells were cocultured with resting splenocytes. Activation of B and T lymphocytes was assessed by measuring cell proliferation, blast formation, and cytokine release. RESULTS: We report that addition of IL-4-treated BMMCs to normal spleen cells resulted within 48 hours in a B- and T-cell activation with substantial amounts of the T(H1) cytokines IFN-gamma and IL-12 and no detectable IL-4. We also demonstrate that mature mast cells in the peritoneal cavity are able to induce spleen cell activation and cytokine release. Addition of antileukocyte function-associated antigen 1 and anti-intercellular adhesion molecule 1 to the cocultures completely abrogates mast cell-induced blast formation and cytokine release. Experiments performed in vivo indicate that spleen cells from mice injected with BMMCs sustain their capacity of proliferation and cytokine production in vitro without any further stimulation. CONCLUSION: These observations suggest that mast cells may exert a helper effect on B and T lymphocytes, initiate T(H1)-type immune responses, and may participate, through this mechanism, in the downregulation of allergic responses.

FcepsilonRI-mediated antigen endocytosis turns interferon-gamma-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-gamma (IFN-gamma) 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 FcepsilonRI-mediated antigen internalization in the regulation of the antigen-presenting function of IFN-gamma-treated mast cells. Here, we report that FcepsilonRI can reverse the IFN-gamma-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-FcepsilonRI engagement which involves tyrosines found in the immunoreceptor tyrosine-based activation motif (ITAM) embedded in the cytoplasmic tail of the FcepsilonRI beta and gamma 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 FcepsilonRI 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.

Expression of functional major histocompatibility complex class II molecules on HMC-1 human mast cells.

Mast cells hold a key position in the defensive mechanisms against exogenous intruders. In this study, we investigated whether human mast cells express functional major histocompatibility complex (MHC) class II molecules that can transduce endogenous signals and present staphylococcal enterotoxin A (SEA) to T cells. Similar to HMC-1 human mast cell line, umbilical cord blood-derived mast cells express HLA-DR, -DP and -DQ molecules on their surface. MHC class II molecules expressed on HMC-1 cells bind significantly the SEA (a natural MHC class II ligand), and their ligation with specific mAbs or with SEA, leads ultrastructural changes, suggesting their degranulation. Recognition of SEA-bound MHC class II molecules on HMC-1 mast cells by the T cell receptor of K25 cells, an SEA-specific murine T cell hybridoma, triggers significant IL-2 secretion by these T cell hybridomas. Hence, our data point out the expression of functional MHC class II molecules on human mast cells, reinforcing the implication of these cells in the defense mechanisms of acquired immunity.

Specific antigen targeting to surface IgE and IgG on mouse bone marrow-derived mast cells enhances efficiency of antigen presentation.

The discovery that bone marrow-derived mast cells can express major histocompatibility complex class II molecules and act as antigen-presenting cells prompted us to evaluate this function when antigen is internalized through fluid-phase endocytosis or via specific uptake by using IgG and IgE antibodies. This study was performed using a specific T-cell hybridoma developed against Lol p 1, the major allergen of grass pollen Lolium perenne. Expression of Fc gamma R and Fc epsilon RI by mast cells led us to investigate the influence of IgG- and IgE-targeted antigen on the antigen-presenting function of mast cells. Internalization of Lol p 1 through different specific IgG monoclonal antibodies (mAb) resulted in the activation of Lol p 1-specific T-cell hybridoma at concentrations about 100-fold less than that required for T-cell stimulation by uncomplexed antigen. IgE-complexed Lol p 1, which facilitates trapping of antigen by mast cells, induced an accelerated and more efficient antigen-presenting capacity of mast cells than that obtained with uncomplexed antigen. However, aggregation of anti-dinitrophenyl (DNP) IgE mAb by the irrelevant antigen DNP-human serum albumin did not substantially increase the capacity of mast cells to present Lol p 1 to T cells. This suggests that the mere aggregation of Fc epsilon RI is not sufficient for enhanced antigen presentation mediated by IgE. Tissue distribution and strategic location of mast cells at the mucosal barriers and their capacity to process the antigen through efficient fluid-phase pinocytosis as well as IgG- and IgE-dependent targeting of antigens provide mast cells with a prominent role in immune surveillance.

IL-4 mRNA transcription is induced in mouse bone marrow-derived mast cells through an MHC class II-dependent signaling pathway.

We have previously shown that mouse bone marrow-derived mast cells (BMMC) can process and present immunogenic peptides to CD4 T cells. Here, we report on a T cell-dependent MHC class II-mediated mast cell activation resulting in IL-4 transcription and protein release. Presentation of optimal doses of ovalbumin peptide 323-339 resulted in IL-2 production by a specific T cell hybridoma and increase in IL-4 mRNA transcription in mast cells. IL-4 mRNA transcription increased by 200-fold in mast cells treated in IL-3/IL-4/granulocyte-macrophage colony-stimulating factor (high presenters) whereas only a tenfold increase or no increase were obtained with IL-3/IL-4/IFN-gamma- or IL-3-treated mast cells (low presenters), respectively. Induction of IL-4 mRNA transcription in purified mast cells by direct ligation of MHC class II molecules, using anti-I-A and anti-I-E-coated beads, indicates that MHC class II molecules are critical in this signaling pathway. However, when compared to T cells, anti-MHC class II-coated beads were less efficient, indicating a potential role of accessory molecules in this mast cell activation process. IgE-independent IL-4 production by mast cells as a result of cognate interaction with CD4 T cells could be critical for the development of type 2 responses. This novel mechanism may contribute to the induction and/or amplification of specific IgE-mediated allergic responses.

Accumulation of major histocompatibility complex class II molecules in mast cell secretory granules and their release upon degranulation.

To investigate the relationship between major histocompatibility complex (MHC) class II compartments, secretory granules, and secretory lysosomes, we analyzed the localization and fate of MHC class II molecules in mast cells. In bone marrow-derived mast cells, the bulk of MHC class II molecules is contained in two distinct compartments, with features of both lysosomal compartments and secretory granules defined by their protein content and their accessibility to endocytic tracers. Type I granules display internal membrane vesicles and are accessed by exogenous molecules after a time lag of 20 min; type II granules are reached by the endocytic tracer later and possess a serotonin-rich electron-dense core surrounded by a multivesicular domain. In these type I and type II granules, MHC class II molecules, mannose-6-phosphate receptors and lysosomal membrane proteins (lamp1 and lamp2) localize to small intralumenal vesicles. These 60-80-nm vesicles are released along with inflammatory mediators during mast cell degranulation triggered by IgE-antigen complexes. These observations emphasize the intimate connection between the endocytic and secretory pathways in cells of the hematopoietic lineage which allows regulated secretion of the contents of secretory lysosomes, including membrane proteins associated with small vesicles.

Functional analysis of Mycoplasma arthritidis-derived mitogen interactions with class II molecules.

The ability of superantigens (SAGs) to trigger various cellular events via major histocompatibility complex (MHC) class II molecules is largely mediated by their mode of interaction. Having two MHC class II binding sites, staphylococcal enterotoxin A (SEA) is able to dimerize MHC class II molecules on the cell surface and consequently induces cytokine gene expression in human monocytes. In contrast, cross-linking with specific monoclonal antibodies or T-cell receptor is required for staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin 1 (TSST-1) to induce similar responses. In the present study, we report how Mycoplasma arthritidis-derived mitogen (MAM) may interact with MHC class II molecules to induce cytokine gene expression in human monocytes. The data presented indicate that MAM-induced cytokine gene expression in human monocytes is Zn2+ dependent. The MAM-induced response is completely abolished by pretreatment with SEA mutants that have lost their capacity to bind either the MHC class II alpha or beta chain, with wild-type SEB, or with wild-type TSST-1, suggesting that MAM induces cytokine gene expression most probably by inducing dimerization of class II molecules. In addition, it seems that SEA and MAM interact with the same or overlapping binding sites on the MHC class II beta chain and, on the other hand, that they bind to the alpha chain most probably through the regions that are involved in SEB and TSST-1 binding.

Exogenous and endogenous antigens are differentially presented by mast cells to CD4+ T lymphocytes.

In the present work, we explored the cytokine-dependent regulation of bone marrow-derived mast cell (BMMC) antigen-presenting cell (APC) function, and co-stimulation requirements, and analyzed the nature of antigens presented to T cells. We observed an up-regulation of the APC function of mast cells induced by granulocyte/macrophage-colony-stimulating factor (GM-CSF) and a complete abrogation by interferon (IFN)-gamma. Expression of co-stimulatory molecules CD80 and CD86 was suggested by the ability of mast cells to activate purified lymph node-derived T cells. Indeed, addition of the fusion protein mCTLA4-Ig strongly inhibited antigen presentation by mast cells to normal T cells and to the T cell hybridoma 3DO-54.8. The regulatory mechanisms of APC function by GM-CSF and IFN-gamma were investigated by measuring CD80 and CD86 transcripts in mast cells. GM-CSF-treated must cells showed a strong increase in the expression of both CD80 and CD86 transcripts, whereas in IFN-gamma-treated mast cells, this expression was completely abrogated. Thus, up- and down-regulation of CD80 and CD86 expression by GM-CSF and IFN-gamma is directly correlated to the APC function. In addition, we analyzed antigen presentation by mast cells of endogenous self-antigens. Mast cells failed to activate anti-I-A or anti-I-E-specific T cell hybridomas and alloreactive T cells in primary mixed lymphocyte reactions (MLR). Furthermore, mast cells did not present the mouse beta 2-microglobulin (m beta 2-m) peptide 25-40, constitutively expressed on B cells. However, mast cells, especially those treated with GM-CSF, activated an anti-m beta 2-m-specific T cell hybridoma in the presence of exogenous peptide. The minor lymphocyte-stimulating antigen-1 Mls-1a is a viral superantigen (vSAG) encoded by the the mouse mammary tumor provirus-7 (MMTV-7). Mast cells, despite a reasonable amount of major histocompatibility complex class II on the cell surface and the presence of MMTV transcripts predicted to encode the vSAG, cannot stimulate in vivo or in vitro V beta 6+ T cells specific for Mls-1a. In contrast, mast cells could present the exogenous bacterial SAG, staphylococcal enterotoxin B (SEB), to specific V beta 8+ T cells. The selective ability of mast cells to present exogenous antigens may have physiological relevance in that mast cells could participate in immune response regulatory mechanisms by discriminating self from nonself.

Mouse bone marrow-derived mast cells and mast cell lines constitutively produce B cell growth and differentiation activities.

The present report describes a novel function of mast cells that consists of a B cell growth activity. The B cell response occurred without any stimulation or preactivation of mast cells. A small number of mast cells was required, since mast cell/B cell ratios as low as 1/100 to 1/10,000 lead to effective B cell activation. Mast cell-dependent B cell activation resulted, within 48 h of incubation, in blast formation, proliferation, and IgM production. Both low and high density B cells were responsive to mast cells. Supernatants from unstimulated mast cells could also activate B cells, suggesting that a B cell-stimulating activity (MC-BSA) is mediated by a soluble factor(s). The addition of anti-IL-4 or anti-IL-6 mAbs or even proteases to the mast cell-derived supernatants did not alter B cell activation. However, treatment of mast cells with mitomycin C or actinomycin D, or paraformaldehyde fixation totally abrogated MC-BSA. Fractionation of mast cell supernatant by gel filtration chromatography resulted in four peaks, ranging from > 200 to 15 kDa, all of which were biologically active on B cells. Because mast cells are known to continuously release proteoglycans, MC-BSA was subjected to chondroitinase and heparinase treatment, but no significant inhibition of B cell activation was obtained. This direct T cell-independent stimulatory effect of mast cells on B cells could account for a mechanism by which plasma cells are continuously produced in lymphoid organs and particularly in bone marrow.

Presentation of soluble antigens by mast cells: upregulation by interleukin-4 and granulocyte/macrophage colony-stimulating factor and downregulation by interferon-gamma.

We recently showed that bone marrow-derived mast cells bore MHC class II molecules and could present antigens to specific T cell hybridomas. This article summarizes the effects of purified recombinant cytokines on the expression of MHC class II molecules by mast cells and on their antigen-presenting capacity. Since IL-3 is essential for mast cell growth, all the cytokines were analyzed in the presence of IL-3. IL-3 downregulated the production of Ia molecules, so that mast cells cultured in IL-3 alone had no antigen presenting ability. In contrast, IL-4 and IFN-gamma upregulated the production of MHC class II molecules, while GM-CSF had no effect. The antigen-presenting capacity of IL-4-treated mast cells was substantially enhanced by incubating these cells with GM-CSF for 2 days. GM-CSF enhanced antigen presentation only in combination with IL-4. The activation of mast cells was reversible and could not be repeated. Finally, incubation of IL-4- or IL-4/GM-CSF-treated mast cells with IFN-gamma led to almost complete inhibition of the antigen-presenting function. These findings provide new insights into the regulation of specific allergic responses.

Modulation of Mycoplasma arthritidis-derived superantigen-induced cytokine gene expression by dexamethasone and interleukin-4.

Activation of human monocytes or monocytic cell lines with all known stimuli coordinately induces the gene expression of various cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and the IL-1 receptor antagonist (IL-1Ra). In contrast, superantigens induce TNF-alpha and IL-1 beta but fail to affect IL-1Ra gene expression, suggesting that activation of monocytes via major histocompatibility complex class II is distinct from other signal transduction pathways. In the present study, we analyzed the regulation of the Mycoplasma arthritidis-derived superantigen (MAM)-induced IL-1 beta and TNF-alpha gene expression by studying the effects of two different anti-inflammatory agents: dexamethasone (DEX) and the T-cell-derived cytokine IL-4. Both agents contributed to the downregulation of MAM-induced IL-1 beta and TNF-alpha gene expression. They accelerated the normal decline of the gene expression of both MAM-induced cytokines by decreasing the stability of mRNAs via the induction or enhanced synthesis of one or more regulatory proteins. In addition, IL-4, but not DEX, induced a strong and rapid expression of IL-1Ra mRNA in MAM-stimulated and unstimulated THP-1 cells in a de novo protein synthesis-independent manner. The capacity of IL-4 to induce IL-1Ra gene expression reinforces its anti-inflammatory activity. This study illustrates some of the mechanisms by which MAM-induced proinflammatory monokine gene expression can be downregulated by IL-4 and DEX.