IL-33/ST2 axis promotes mast cell survival via BCLXL

Mast cells (MC) are potent innate immune cells that accumulate in chronically inflamed tissues. MC express the IL-33 receptor IL-1 receptor-related protein ST2 at high level, and this IL-1 family cytokine both activates MC directly and primes them to respond to other proinflammatory signals. Whether IL-33 and ST2 play a role in MC survival remains to be defined. In skin-derived human MC, we found that IL-33 attenuated MC apoptosis without altering proliferation, an effect mediated principally through the antiapoptotic molecule B-cell lymphoma-X large (BCLXL). Murine MC demonstrated a similar mechanism, dependent entirely on ST2. In line with these observations, St2-/- mice exhibited reduced numbers of tissue MC in inflamed arthritic joints, in helminth-infected intestine, and in normal peritoneum. To confirm an MC-intrinsic role for ST2 in vivo, we performed peritoneal transfer of WT and St2-/- MC. In St2-/- hosts treated with IL-33 and in WT hosts subjected to thioglycollate peritonitis, WT MC displayed a clear survival advantage over coengraftedSt2-/- MC. IL-33 blockade specifically attenuated this survival advantage, confirming IL-33 as the relevant ST2 ligand mediating MC survival in vivo. Together, these data reveal a cell-intrinsic role for the IL-33/ST2 axis in the regulation of apoptosis in MC, identifying thereby a previously unappreciated pathway supporting expansion of the MC population with inflammation

Negative regulation of FceRI signaling by FcgRII costimulation in human blood basophils

Background: Signaling through the antigen receptors of human B and T cells and the high-affinity IgE receptor Fc-epsilonRI of rodent mast cells is decreased by cross-linking these receptors to the low-affinity IgG receptor Fc-gammaRIIB. The inhibition is thought to involve the tyrosine phosphorylationof immunoreceptor tyrosine–based inhibitory motifs (ITIMs) in the Fc-gammaRII 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 Fc-epsilonRI signaling generates mediators and cytokines responsible for allergic inflammation. We proposed to determine whether Fc-epsilonRI signaling is inhibited by Fc-gammaRII costimulation in human basophils and to explore the underlying mechanism as an approach to improving the treatment of allergic inflammation. Methods: Fc-gammaR expression on human basophils was examined using flow cytometryand RT-PCR analysis. Fc-gammaRII/Fc-epsilonRI 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 Fc-gammaRIIB ITIM sequence were used for adsorption assays. Results: We report that peripheral blood basophils express Fc-gammaRII (in both the ITIM-containing Fc-gammaRIIB and the immunoreceptor tyrosine–based activation motif–containing Fc-gammaRIIA forms) and that costimulating Fc-gammaRII and Fc-epsilonRI inhibits basophil Fc-epsilonRI-mediated histamine release, IL-4 production, and Ca2+ mobilization. The inhibition of basophil Fc-epsilonRI signaling by Fc-gammaRII/Fc-epsilonRI costimulation is linked to a significant decrease in Syk tyrosine phosphorylation. Human basophils express all 3 SH2-containing phosphatases. Conclusions: Evidence that Fc-gammaRII/Fc-epsilonRI costimulation induces SHP-1 translocation from the cytosolic to membrane fractions of basophils and that biotinylated synthetic peptides corresponding to the phosphorylated Fc-gammaRIIB ITIM sequence specifically recruit SHP-1 from basophil lysates particularly implicates this protein phosphatase in the negative regulation of Fc-epsilonRI signaling by costimulated Fc-gammaRII

Purification of human basophils by density and size alone

Basophils typically account for approximately 1% of the white cells in peripheral blood. We have developed a unique method for purifying basophils from whole blood of normal subjects to at least 95% purity. Basophils are separated from other cell types dy density-dependent sedimentation in Percoll and cell sorting, based solely on their size and granularity. The mean overall yield ranged from 5% to 28%. The procedure is typically completed within 4 h. The highly purified basophils obtained are functionally competent and morphologically intact. They release histamine in response to FceRI-mediated stimulation, express FceRI and BSP-1 ligand as analyzed by flow cytometry, and exhibit the known characteristic ultrastructural features of basophils by electron microscopy. This procedure avoids positive-selection antibodies that might perturb receptors on basophils or negative-selection antibodies that might activate other cell types, and can be used to obtain basophils for studies in vitro

Inhibition of allergen-specific IgE reactivity by a human Ig Fcgamma-Fcepsilon bifunctional fusion protein

Background: Coaggregating FceRI with FcgRII receptors holds great potential for treatment of IgE-mediated disease by inhibiting FceRI signaling. We have previously shown that an Fcg-Fce fusion protein, human IgG-IgE Fc fusion protein (GE2), could inhibit FceRI-mediated mediator releases in vitro and in vivo. Objective: We sought to test whether GE2 was capable of blocking mediator release from FceRI cells sensitized with IgE in vivo or in vitro before exposure to GE2, a critical feature for GE2 to be clinically applicable.Methods: GE2 was tested for its ability to inhibit Fel d 1–induced mediator release from human blood basophils from subjects with cat allergy, human lung-derived mast cells, human FceRIa transgenic mice sensitized with human cat allergic serum, and rhesus monkeys naturally allergic to the dust mite Dermatophagoides farinae. Results: Basophils from subjects with cat allergy and lung mast cells degranulate when challenged with Fel d 1 and anti-IgE, respectively. GE2 itself did not induce mediator release but strongly blocked this Fel d 1– and anti-IgE–driven mediator release. GE2 was able to block Fel d 1–driven passive cutaneous anaphylaxis at skin sites sensitized with human serum from subjects with cat allergy in human FceRIa transgenic mice, but by itself, GE2 did not induce a passive cutaneous anaphylaxis reaction. Finally, GE2 markedly inhibited skin test reactivity to D farinae in monkeys naturally allergic to this allergen, with complete inhibition being observed at 125 ng. Conclusion: GE2 is able to successfully compete for FceRs and FcgRs on cells presensitized in vitro and in vivo and lead to inhibition of IgE-mediated reactivity through coaggregation of FceRI with FcgRII

Fullerenes in Medicine; Will it ever Occur?

Fullerenes represent a group of compounds having unique properties that make them attractivecandidates for use as a platform for developing new medical applications. The carbon cage(usually C60 and C70) of empty cage fullerenes (Figure 1-left/middle) are being developed astherapeutics for disease processes such as multiple sclerosis, neurodegeneration, HIV infection,cancer, radiation exposure, ischemia, allergic disease, infectious disease, and generalinflammation. Metallo-fullerenes (Figure 1-right; that have metals enclosed within the carboncage) are being developed into new biomarker homing, diagnostic contrast agents for MRI.Lastly, a new class of theranostics are being developed that combine cell targetingcapabilities/imaging with a therapeutic payload. Their inherent properties combined with theirability to be derivatized with side chains results in almost limitless new chemical structuresmaking them ideal platform molecules for new solutions to basic biological problems. However,one of the biggest obstacles that have kept this class of compounds from potentially improvinghuman health and reducing health care costs is the concern about toxicity. This has been due, inpart, to the lack of standard structural relationships that affect biological outcomes of FullereneDerivatives (FD)

Antigen-induced reduction in mast cell and basophil functional responses due to reduced Syk protein levels

Background: The high-affinity IgE receptor, FceRI, is unresponsive on mast cells and basophils from people in several populations through an unknown mechanism. Similarly, FceRI-positive basophils from ‘nonreleasers’ are IgE-unresponsive and are deficient in the tyrosine kinase Syk. Objective: To test the hypothesis that cross-linking FceRI on mast cells and basophils leads to FceRI nonresponsiveness through reduction in Syk protein levels. Methods: Human mast cells and basophils were used to determine if FceRI hyporesponsiveness correlated with reduced Syk levels. Results: It is shown that suboptimal antigen challenge, that did not lead to significant mediator release, induced nonresponsiveness and correlated with reduced Syk. Other IgE-associated signaling molecules were unaffected by the same treatment. The ability of IgE-unresponsive mast cells to regain FceRI responsiveness is paralleled by increased cellular Syk levels in vitro. The reduction of Syk levels with suboptimal antigen concentrations was calcium independent and mediated through a proteasome-dependent mechanism. Conclusion: These findings confirm and extend our knowledge about a novel regulatory mechanism for maintaining FceRI in a quiescent state. This mechanism may also explain why low concentrations of allergen given to patients during allergen immunotherapy induce FceRI nonresponsiveness and therapeutic benefit without inducing systemic anaphylaxis

Environmental polycyclic aromatic hydrocarbons, benzo(a) pyrene (BaP) and BaP-quinones, enhance IgE-mediated histamine release and IL-4 production in human basophils

Polycyclic aromatic hydrocarbons (PAHs) are major components of diesel exhaust particles found in pollutant respirable particles. There is growing evidence that these fossil fuel combustion products exacerbate allergic inflammation. Basophils contribute to allergic inflammation through the release of preformed and granule-derived mediators. To determine whether allergens and PAHs interact, we incubated human basophils with PAHs and measured the release of histamine and IL-4 with and without added antigen. None of the PAHs induced mediator release by itself and none affected total cellular histamine levels. However, several PAHs enhanced histamine release and IL-4 production in response to crosslinking the high-affinity IgE receptor, FceRI. The enhancement seen with 1,6-BaP-quinone involved an increase in tyrosine phosphorylation in several different substrates, including the FceRI-associated tyrosine kinase, Lyn, and elevated reactive oxygen species (ROS) levels detected by dichlorofluorescein fluorescence and flow cytometry. The PAH-induced enhancement of mediator release and ROS production could be inhibited with the antioxidant N-acetylcysteine. These data provide further evidence that environmental pollutants can influence allergic inflammation through enhanced FceRI-coupled mediator release from human basophils

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 of the FceRI-activated tyrosine kinases Lyn, Syk, and Zap-70 in human basophils

Background: In human blood basophils, cross-linking the high-affinity IgEreceptor FceRI 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 FceRI signaling was explored by using human basophils isolated by Percoll gradient centrifugation followed by negative and/or positive selection with antibody-coated magnetic beads. FceRI cross-linking of more than 95% pure basophil preparations activates the protein-tyrosine kinases Lyn and Syk, previously linked to FceRI-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 FceRI-mediated signaling in human basophils

Polyhydroxylated C60 fullerene (fullerenol) attenuates neutrophilic lung inflammation in mice

Inflammation is crucial to eliminate pathogens and promote repair of injured tissue. However, excessive or persistent inflammation can contribute to tissue injury and the pathogenesis and exacerbation of diseases, including inflammatory lung diseases, such as chronic obstructive pulmonary disease [1] and silicosis [2]. Neutrophilic inflammation is an important aspect of chronic obstructive pulmonary disease [1, 3, 4] and silicosis [2, 5]. Thus, in human beings, a relationship between exposure to respirable silica in coal mine dust and pulmonary inflammation is seen, resulting in an elevated neutrophil count in bronchoalveolar lavage fluid (BALF) [6]. Exposure to silica can cause silicosis, where the severe inflammation in the lung appears to be an initiating step in the development of the disease [7]. The quartz particles can in itself generate reactive oxygen species (ROS), but additional inflammatory injuries appear to be a result of the influx of inflammatory cells [2]. The cell-generated ROS and nitric oxide radicals are hallmarks of the toxicity of the quartz particles [8, 9], and quartz-induced inflammation is characterised by, for example, neutrophilic inflammation in rodents [10]. The importance of neutrophils in the development of inflammatory lung diseases has been reported in rodents, where exposure to quartz resulted in induced influx of neutrophils [2, 4, 11-15]. Furthermore, it was shown that treatment with anti-macrophage inflammatory protein 2 (MIP-2) antiserum prior to quartz exposure attenuated neutrophil influx [16], suggesting that MIP-2 can play an important role in quartz-induced neutrophilic lung inflammation