A potential new target for asthma therapy: a disintegrin and metalloprotease 10 (ADAM10) involvement in murine experimental asthma.

BACKGROUND: Elevated levels of CD23, a natural regulator of IgE production, have been shown to decrease the signs of lung inflammation in mice. The aim of this study was to study the involvement of ADAM10, the primary CD23 sheddase, in experimental asthma. METHODS: ADAM10 was blocked either by using mice with a B-cell-specific deletion of the protease or pharmacologically by intranasal administration of selective ADAM10 inhibitors. Airway hypersensitivity (AHR) and bronchoaveolar lavage fluid (BALF) eosinophilia and select BALF cytokine/chemokine levels were then determined. RESULTS: Using an IgE and mast cell-dependent mouse model, B-cell-specific ADAM10(-/-) mice (C57B/6 background) exhibited decreased eosinophilia and AHR when compared with littermate (LM) controls. Treatment of C57B/6 mice with selective inhibitors of ADAM10 resulted in an even further decrease in BALF eosinophilia, as compared with the ADAM10(-/-) animals. Even in the Th2 selective strain, Balb/c, BALF eosinophilia was reduced from 60% to 23% respectively. In contrast, when an IgE/mast cell-independent model of lung inflammation was used, the B-cell ADAM10(-/-) animals and ADAM10 inhibitor treated animals had lung inflammation levels that were similar to the controls. CONCLUSIONS: These results thus show that ADAM10 is important in the progression of IgE-dependent lung inflammation. The use of the inhibitor further suggested that ADAM10 was important for maintaining Th2 levels in the lung. These results thus suggest that decreasing ADAM10 activity could be beneficial in controlling asthma and possibly other IgE-dependent diseases.

Regulation of the very late antigen-4-mediated adhesive activity of normal and nonreleaser basophils: roles for Src, Syk, and phosphatidylinositol 3-kinase.

Normal human basophils express the integrin, VLA-4, and cross-linking their high-affinity IgE receptor, FcepsilonRI, increases their VLA-4-dependent adhesion to VCAM-1-transfected Chinese hamster ovary (CHO) cells. Here we show that the FcepsilonRI-mediated up-regulation of normal basophil VLA-4 adhesion is abolished by the Src inhibitor, PP1, the Syk inhibitor, ER-27319, and the phosphatidylinositol 3-kinase inhibitor, wortmannin. PP1, but not ER-27319 or wortmannin, also reduces basal adhesion and adhesion stimulated by chemotactic peptide, by Ca(++) ionophores, and by phorbol myristate acetate (PMA). Nonreleaser basophils (the consistently Syk-deficient, variably Lyn-deficient, severely degranulation-impaired cells found in about 10% of donors) share the PP1 phenotype of lowered basal adhesion, no FcepsilonRI-mediated adhesion up-regulation, and reduced adhesive responses to chemoattractant ionophores and PMA. These results implicate Src kinases in the control of basal VLA-4 activity and place Syk and phosphatidylinositol 3-kinase in the pathway linking FcepsilonRI cross-linking to VLA-4 up-regulation. Both Src and Syk-regulated components of adhesion may be impaired in nonreleaser basophils.

The identification and characterization of umbilical cord blood-derived human basophils.

Cross-linking allergen-specific immunoglobin E on human peripheral blood basophils results in the release of histamine and other inflammatory mediators that initiate allergy and asthma. The signaling pathways leading from IgE binding to mediator release have not been well established, mainly due to the difficulty in obtaining adequate numbers of highly purified basophils. It was the goal of this study to easily obtain Fc epsilonRI-positive human basophils in high yield and purity for studies of signal transduction pathways. We describe an in vitro culture system in which pulsing normal human cord blood leukocytes with interleukin-3 (IL-3) for 3-4 h followed by incubation in medium with fetal bovine serum generates a cell population that is predominately Fc epsilonRI positive between 14 and 28 days of culture. These cells resemble peripheral blood basophils when examined by light and electron microscopy. Like normal blood basophils, they express the integrins, CD11b, CD18, CD29, and CD49d. A majority of the IL-3-pulsed cells also express a marker recognized by the basophil-specific antibody, 2D7. Fc epsilonRI cross-linking results in a time and dose-dependent release of histamine. Fc epsilonRI cross-linking also stimulates protein-tyrosine phosphorylation, thought to be the first event leading to the IgE-mediated activation of peripheral blood basophils. These studies establish cord blood as an accessible source from which basophil-like cells can be developed to examine Fc epsilonRI-mediated signal transduction.

Detection of MCT and MCTC types of human mast cells by immunohistochemistry using new monoclonal anti-tryptase and anti-chymase antibodies.

We developed an improved immunohistochemical technique for distinguishing human mast cells of the MCT (tryptase-positive, chymase-negative) and MCTC (tryptase-positive, chymase-positive) types utilizing a biotinylated murine anti-chymase monoclonal antibody (MAb), termed B7, and an alkaline phosphatase-conjugated murine anti-tryptase MAb, termed G3. The B7 MAb also was used to show the selective presence of chymase in mast cells. The distribution of MCT and MCTC cells in Carnoy's fluid-fixed tissue sections of human lung, skin, small intestine, and tonsils was analyzed by the new technique and the results compared to those obtained with the older method using a rabbit polyclonal antichymase antibody and a mouse anti-tryptase MAb in indirect immunoperoxidase and indirect immunoalkaline phosphatase protocols, respectively. In tissues known to contain predominantly mature mast cells, there were no quantitative differences between the two techniques, although the staining intensity achieved with the anti-chymase MAb was greater and without development of high background, compared to results achieved with the polyclonal antibody. MCT cells were the predominant type seen in the alveoli of the lung (93%) and in the small intestinal mucosa (81%). MCTC cells predominanted in the skin (99%) and in the small intestinal submucosa (77%) and, to a lesser degree, in tonsils (60%). However, in newborn foreskin tissue which contains predominantly immature forms of mast cells, 75% of all mast cells were stained uniformly and intensely with B7, whereas only 43% were stained with the polyclonal anti-chymase antibody. Therefore, the use of MAb provides for better standardization of reagents and more accurate assessment of the distribution of human MCT and MCTC cells in tissues than previously available methods.

Identification and partial characterization of a unique marker for human basophils.

A unique marker for human basophils is needed to precisely determine the involvement of this cell type in clinical disease. To search for a marker of the basophil secretory granule, mouse hybridomas were generated against purified human basophils and screened for basophil-selective Ab. One hybridoma (2D7) produced an IgG1 kappa Ab that labeled basophils, but not lymphocytes, monocytes, eosinophils, neutrophils, and mast cells by an indirect immunoperoxidase procedure. The pattern of basophil staining was cytoplasmic and granular by light microscopy. By immunogold electron microscopy, the 2D7 ligand was localized to secretory granules. Activated basophils showed reduced 2D7-dependent staining intensity, consistent with a secretory granule localization. Tissue sections of normal skin, lung, and bowel showed no reactivity with 2D7, consistent with the anticipated absence of basophils in these tissues. 2D7 staining of basophils was clearly distinct from metachromatic staining, which was presumably dependent on proteoglycan. Extracts of normal human basophils subjected to Western blotting with 2D7 exhibited two predominant bands at apparent molecular masses of 76,150 and 72,260 Da. In summary, the 2D7 ligand appears to be a specific marker for human basophils and may facilitate the assessment of basophil involvement in diseases such as asthma, anaphylaxis, and atopic dermatitis.

Identification of the Fc epsilonRI-activated tyrosine kinases Lyn, Syk, and Zap-70 in human basophils.

BACKGROUND: In human blood basophils, cross-linking the high-affinity IgE receptor Fc epsilonRI with multivalent antigen activates a signaling pathway leading to Ca2+ mobilization, actin polymerization, shape changes, secretion, and cytokine production. METHODS AND RESULTS: The role of tyrosine kinases in human Fc epsilonRI signaling was explored by using human basophils isolated by Percoll gradient centrifugation followed by negative and/or positive selection with antibody-coated magnetic beads. Fc epsilonRI cross-linking of more than 95% pure basophil preparations activates the protein-tyrosine kinases Lyn and Syk, previously linked to Fc epsilonRI-coupled rodent mast cell activation, as well as Zap-70, previously implicated in T-cell receptor signaling, and causes the tyrosine phosphorylation of multiple proteins. The presence of Lyn, Syk, and Zap-70 in basophils was confirmed by Western blotting in lysates of highly purified basophils and independently by confocal fluorescence microscopy in cells labeled simultaneously with kinase-specific antibodies and with the basophil-specific antibody 2D7. Comparable amounts of Lyn and Syk were found in basophils and B cells, whereas T cells appear to have greater amounts of Zap-70 than basophils. The tyrosine kinase inhibitor piceatannol spares IgE-mediated Lyn activation but inhibits IgE-induced Syk and Zap-70 activation as well as overall protein tyrosine phosphorylation and secretion. Overall protein-tyrosine phosphorylation increases steadily over a range of anti-IgE concentrations that are low to optimal for secretion. However, tyrosine phosphorylation continues to increase at high anti-IgE concentrations that elicit very little secretion (the characteristic high-dose inhibition of secretion). CONCLUSIONS: Our data demonstrate the association of anti-IgE-stimulated, protein-tyrosine phosphorylation by a cascade of tyrosine kinases, including Zap-70 as well as Lyn and Syk, with the initiation of Fc epsilonRI-mediated signaling in human basophils.

Immunohistochemical detection of human basophils in postmortem cases of fatal asthma.

The role of human basophils in bronchial asthma has been hard to define. In this study, we used the basophil-specific monoclonal antibody (mAb), 2D7, in postmortem lung sections from individuals who die in status asthmaticus (fatal asthma [FA]) to determine if the pathology of FA is associated with an increase in basophil numbers in the lung. As controls, we used lung sections of patients who had a history of asthma but died from nonasthmatic causes (nonfatal asthma [NFA]) as well as patients with no history of asthma (control [C]). In lung sections from all three groups, basophils were scattered throughout the large and small airways, airway epithelium, submucosa, and alveolar walls. The numbers of basophils in the lungs of patients with FA ranged from 41 to 119 cells/mm(2), significantly more than the numbers of basophils in lungs from individuals with a history of asthma (NFA; 0 to 16 cells/ mm(2)) and in the control lungs (C; 0 to 13 cells/mm(2)). In contrast, CD45-positive cells were not significantly different in the airways of FA and NFA, although there were significant increases in the two groups compared with control subjects. In summary, basophil infiltration was significantly increased in lungs from individuals who died from asthma, supporting the hypothesis that basophils are involved in the pathogenesis of FA.

Syk deficiency in nonreleaser basophils.

BACKGROUND: Peripheral blood basophils from 10% to 20% of donors fail to degranulate in response to cross-linking the high-affinity IgE receptor FcepsilonRI. The molecular mechanisms underlying the nonreleaser phenotype have not been established. OBJECTIVES: Our aim was to compare the expression of FcepsilonRI-associated protein tyrosine kinases between nonreleaser and releaser basophils. METHODS: With use of Western blotting we investigated Syk and Lyn protein levels in highly purified basophils from 3 anti-IgE nonreleasers and 2 releasers. RESULTS: We identified 3 healthy nonatopic donors whose nonreleaser basophils express FcepsilonRI normally but fail to express protein for the tyrosine kinase Syk, which is implicated in the initiation of FcepsilonRI-mediated secretion. Protein levels for the tyrosine kinase Lyn are somewhat reduced but not absent in nonreleaser basophils. Levels of Lyn and Syk protein are similar in B cells, eosinophils, and neutrophils from releaser and nonreleaser donors. During these studies one nonreleaser "converted" into a releaser with concomitant basophil Syk expression. CONCLUSION: The absence of detectable Syk could explain the nonreleaser phenotype of basophils from some donors.

Multiple defects in Fc epsilon RI signaling in Syk-deficient nonreleaser basophils and IL-3-induced recovery of Syk expression and secretion.

Human basophils respond to Ag-induced cross-linking of their high affinity IgE receptor, FcepsilonRI, by releasing histamine and other mediators from granules, producing IL-4 and other cytokines and, as shown in this study, by forming membrane ruffles and showing increased very late Ag-4 (VLA-4)-mediated adhesion to VCAM-1-expressing target cells. We have identified five blood donors whose basophils lack detectable levels of the FcepsilonRI-associated protein tyrosine kinase, Syk. Despite showing no obvious ultrastructural differences from normal basophils, nonreleaser basophils fail to form membrane ruffles, to show increased VLA-4-mediated adhesive activity, or to produce IL-4 in response to FcepsilonRI cross-linking. Although Syk protein levels are suppressed in basophils from all five donors, Syk mRNA is consistently present. Furthermore, culturing nonreleaser basophils for 4 days with IL-3 restores Syk protein expression and FcepsilonRI-mediated histamine release. Understanding the reversible suppression of Syk protein expression in nonreleaser basophils, and learning to replicate this property in patients with allergic inflammation could be a powerful and specific way to limit symptomatic disease.

Immunologically mediated signaling in basophils and mast cells: finding therapeutic targets for allergic diseases in the human FcvarepsilonR1 signaling pathway.

The high affinity IgE receptor, FcvarepsilonRI, plays key roles in an array of acute and chronic human allergic reactions including asthma, allergic rhinitis, atopic dermatitis, urticaria and anaphylaxis. In humans and rodents, this receptor is found at high levels on basophils and mast cells where its activation by IgE and multivalent antigen produces mediators and cytokines responsible for FcvarepsilonRI-dependent acute inflammation. Mast cells can additionally contribute to sustained inflammatory responses by internalizing antigen bound to IgE-FcvarepsilonRI complexes for processing to peptides and presentation to T cells. In humans, the FcvarepsilonRI is also expressed, at lower density, on monocytes, macrophages and dendritic cells (DC) where its likely functions again include both signaling to mediator and cytokine production and antigen presentation. Our laboratories have focused on defining the earliest steps in the FcvarepsilonRI signaling cascade in basophils and mast cells and on developing new routes to control allergic inflammation based on inhibiting these events. Here, we describe novel strategies to limit antigen-stimulated FcvarepsilonRI signaling by: (1) sequestering the FcvarepsilonRI-associated protein-tyrosine kinase, Lyn, that initiates FcvarepsilonRI signaling; (2) eliminating; or (3) inactivating the protein-tyrosine kinase, Syk, that propagates FcvarepsilonRI signaling; and (4) establishing inhibitory crosstalk between FcvarepsilonRI and a co-expressed receptor, FcgammaRII, that again limits FcvarepsilonRI-mediated Syk activation. These strategies may form the basis for new therapies for allergic inflammation.

Quantitation of tryptase, chymase, Fc epsilon RI alpha, and Fc epsilon RI gamma mRNAs in human mast cells and basophils by competitive reverse transcription-polymerase chain reaction.

Competitive reverse transcription-PCR assays developed for human tryptase, chymase, Fc epsilon RI alpha, and Fc epsilon RI gamma mRNA molecules were applied to the HMC-1 leukemic mast cell line, the KU812 leukemic basophil cell line, mast cells dispersed from lung and skin, and peripheral blood basophils. Relative amounts of alpha-tryptase and beta-tryptase mRNA were determined by analysis of BseAI digests of PCR products. Tryptase expression was highest in tissue-derived mast cells, lowest in basophils and KU812 cells, and intermediate in HMC-1 cells. beta-Tryptase mRNA predominated in HMC-1 and KU812 cells; mixtures of alpha- and beta-tryptase were found in tissue mast cells; and alpha-tryptase predominated in basophils. Chymase mRNA was more abundant in skin-derived (nearly all of the MCTC type) than lung-derived (variable amounts of MCTC and MCT cells) mast cells. Small amounts of chymase mRNA were detected in HMC-1 cells; none was found in basophils, in KU812 cells, or in the one preparation of 100% MCT cells derived from lung. Comparable amounts of Fc epsilon RI alpha and Fc epsilon RI gamma mRNA molecules were measured in basophils and tissue-derived mast cells, lesser amounts were detected in KU812 cells, and almost none was detected in HMC-1 cells. Thus, steady state levels of the granule and membrane resident molecules examined in our study are transcriptionally regulated in mast cells and basophils.

Immunoelectron microscopic localization of galectin-3, an IgE binding protein, in human mast cells and basophils.

Galectin-3 is an endogenous soluble lectin within the family called galectins that bind beta-galactosides. Homologs of the protein isolated from different sources were previously designated as IgE-binding protein (epsilon BP), CBP35, CPB30, Mac-2, RL-29, RLL, L-29, and HL-29. All are now renamed galectin-3. This lectin is widely distributed in cells and tissues of mice, rats, dogs, hamsters, and humans. Light microscopic immunohistochemistry and ultrastructural immunogold labeling methods were used to determine the distribution of galectin-3 in human mast cells of several organs, in mast cells developed in vitro from human fetal liver cells, and in human peripheral blood basophils. Immunolabeling for the protein was observed in mast cells from all sources and in basophils. The lectin was detected in the nucleus and/or the cytoplasm. The nuclear labeling was over heterochromatin whereas euchromatin was unlabeled. Cytoplasmic labeling was concentrated over secretory granules. The intensity of staining generally was greater in mast cells of skin when compared with that of mast cells in other locations and with that of basophils. Studies have indicated that in mast cells galectin-3 may be involved in promoting their adhesion to basal laminae. In this study the localization of galectin-3 in the secretory granules of human mast cells and basophils suggests that these cells may release this lectin when activated to degranulate.

Negative regulation of FcepsilonRI signaling by FcgammaRII costimulation in human blood basophils.

BACKGROUND: Signaling through the antigen receptors of human B and T cells and the high-affinity IgE receptor FcepsilonRI of rodent mast cells is decreased by cross-linking these receptors to the low-affinity IgG receptor FcgammaRII. The inhibition is thought to involve the tyrosine phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the FcgammaRIIB cytoplasmic tail, creating binding sites for SH2-containing protein (Src homology domain containing protein tyrosine phosphatase 1 and 2 [SHP-1, SHP-2]) and/or lipid (SH2 domain-containing polyphosphatidyl-inositol 5-phosphatase) phosphatases that oppose activating signals from the costimulated antigen receptors. OBJECTIVE: In human basophils and mast cells FcepsilonRI signaling generates mediators and cytokines responsible for allergic inflammation. We proposed to determine whether FcepsilonRI signaling is inhibited by FcgammaRII costimulation in human basophils and to explore the underlying mechanism as an approach to improving the treatment of allergic inflammation. METHODS: FcgammaR expression on human basophils was examined using flow cytometry and RT-PCR analysis. FcgammaRII/FcepsilonRI costimulation was typically accomplished by priming cells with anti-dinitrophenol (DNP) IgE and anti-DNP IgG and stimulating with DNP-BSA. Phosphatases were identified by Western blotting, and their partitioning between membrane and cytosol was determined by cell fractionation. Biotinylated synthetic peptides and phosphopeptides corresponding to the FcgammaRIIB ITIM sequence were used for adsorption assays. RESULTS: We report that peripheral blood basophils express FcgammaRII (in both the ITIM-containing FcgammaRIIB and the immunoreceptor tyrosine-based activation motif-containing FcgammaRIIA forms) and that costimulating FcgammaRII and FcepsilonRI inhibits basophil FcepsilonRI-mediated histamine release, IL-4 production, and Ca(2+) mobilization. The inhibition of basophil FcepsilonRI signaling by FcgammaRII/FcepsilonRI costimulation is linked to a significant decrease in Syk tyrosine phosphorylation. Human basophils express all 3 SH2-containing phosphatases. CONCLUSIONS: Evidence that FcgammaRII/FcepsilonRI costimulation induces SHP-1 translocation from the cytosolic to membrane fractions of basophils and that biotinylated synthetic peptides corresponding to the phosphorylated FcgammaRIIB ITIM sequence specifically recruit SHP-1 from basophil lysates particularly implicates this protein phosphatase in the negative regulation of FcepsilonRI signaling by costimulated FcgammaRII.

Real time analysis of the affinity regulation of alpha 4-integrin. The physiologically activated receptor is intermediate in affinity between resting and Mn(2+) or antibody activation.

This work examines the affinity of alpha(4)beta(1)-integrin and whether affinity regulation by G protein-coupled receptor (GPCR) and chemokines receptors is compatible with cell adhesion mediated between alpha(4)-integrin and vascular cell adhesion molecule-1. We used flow cytometry to examine the binding of a fluorescent derivative of an LDV peptide (Chen, L. L., Whitty, A., Lobb, R. R., Adams, S. P., and Pepinsky, R. B. (1999) J. Biol. Chem. 274, 13167-13175) to several cell lines and leukocytes with alpha(4)-integrin ranging from about 2,000 to 100,000 sites/cell. The results support the idea that alpha(4)-integrins exhibit multiple affinities and that affinity changes are regulated by the dissociation rate and conformation. The affinity varies by 3 orders of magnitude with the affinity induced by binding mAb TS2/16 plus Mn(2+) > Mn(2+) ' TS2/16 > activation because of occupancy of GPCR or chemokines receptor > resting receptors. A significant fraction of the receptors respond to the activating process. The change in alpha(4)-integrin affinity and the corresponding change in off rates mediated by GPCR receptor activation are rapid and transient, and their duration depends on GPCR desensitization. The affinity changes mediated by IgE receptor or interleukin-5 receptor persist longer. It appears that the physiologically active state of the alpha(4)-integrin, determined by inside-out signaling, has similar affinity in several cell types.