Sphingosine kinase 1 is pivotal for Fc epsilon RI-mediated mast cell signaling and functional responses in vitro and in vivo

Mast cell degranulation is pivotal to allergic diseases; investigating novel pathways triggering mast cell degranulation would undoubtedly have important therapeutic potential. FcepsilonRI-mediated degranulation has contradictorily been shown to require SphK1 or SphK2, depending on the reports. We investigated the in vitro and in vivo specific role(s) of SphK1 and SphK2 in FcepsilonRI-mediated responses, using specific small interfering RNA-gene silencing. The small interfering RNA-knockdown of SphK1 in mast cells inhibited several signaling mechanisms and effector functions, triggered by FcepsilonRI stimulation including: Ca(2+) signals, NFkappaB activation, degranulation, cytokine/chemokine, and eicosanoid production, whereas silencing SphK2 had no effect at all. Moreover, silencing SPHK1 in vivo, in different strains of mice, strongly inhibited mast cell-mediated anaphylaxis, including inhibition of vascular permeability, tissue mast cell degranulation, changes in temperature, and serum histamine and cytokine levels, whereas silencing SPHK2 had no effect and the mice developed anaphylaxis. Our data differ from a recent report using SPHK1(-/-) and SPHK2(-/-) mice, which showed that SphK2 was required for FcepsilonRI-mediated mast cell responses. We performed experiments in mast cells derived from SPHK1(-/-) and SPHK2(-/-) mice and show that the calcium response and degranulation, triggered by FcepsilonRI-cross-linking, is not different from that triggered in wild-type cells. Moreover, IgE-mediated anaphylaxis in the knockout mice showed similar levels in temperature changes and serum histamine to that from wild-type mice, indicating that there was no protection from anaphylaxis for either knockout mice. Thus, our data strongly suggest a previously unrecognized compensatory mechanism in the knockout mice, and establishes a role for SphK1 in IgE-mediated mast cell responses.

The basophil activation test in immediate-type drug allergy

Diagnosis of drug allergy involves first the recognition of sometimes unusual symptoms as drug allergy and, second, the identification of the eliciting drug. This is an often difficult task, as the clinical picture and underlying pathomechanisms are heterogeneous. In clinical routine, physicians frequently have to rely upon a suggestive history and eventual provocation tests, both having their specific limitations. For this reason both in vivo (skin tests) and in vitro tests are investigated intensively as tools to identify the disease-eliciting drug. One of the tests evaluated in drug allergy is the basophil activation test (BAT). Basophils with their high-affinity IgE receptors are easily accessible and therefore can be used as indicator cells for IgE-mediated reactions. Upon allergen challenge and cross-linking of membrane-bound IgE antibodies (via Fc-epsilon-RI) basophils up-regulate certain activation markers on their surface such as CD63 and CD203c, as well as intracellular markers (eg, phosphorylated p38MAPK). In BAT, these alterations can be detected rapidly on a single-cell basis by multicolor flow cytometry using specific monoclonal antibodies. Combining this technique with in vitro passive sensitization of donor basophils with patients' serum, one can prove the IgE dependence of a drug reaction. This article summarizes the authors' current experience with the BAT in the diagnostic management of immediate-type drug allergy mediated by drug-specific IgE antibodies.

Expression and biological activities of bovine interleukin 4: effects of recombinant bovine interleukin 4 on T cell proliferation and B cell differentiation and proliferation in vitro

Interleukin 4 (IL-4) is a pleotropic cytokine affecting a wide range of cell types in both the mouse and the human. These activities include regulation of the growth and differentiation of both T and B lymphocytes. The activities of IL-4 in nonprimate, nonmurine systems are not well established. Herein, we demonstrate in the bovine system that IL-4 upregulates production of IgM, IgG1, and IgE in the presence of a variety of costimulators including anti-IgM, Staphylococcus aureus cowan strain I, and pokeweed mitogen. IgE responses are potentiated by the addition of IL-2 to IL-4. Culture of bovine B lymphocytes with IL-4 in the absence of additional costimulators resulted in the increased surface expression of CD23 (low-affinity Fc epsilon RII), IgM, IL-2R, and MHC class II in a dose-dependent manner. IL-4 alone increased basal levels of proliferation of bulk peripheral blood mononuclear cells but in the presence of Con A inhibited proliferation. In contrast to the activities of IL-4 in the murine system, proliferation of TH1- and TH2-like clones was inhibited in a dose-dependent manner as assessed by antigen-or IL-2-driven in vitro proliferative responses. These observations are consistent with the role of IL-4 as a key player in regulation of both T and B cell responses.

Human IgA Fc receptor FcαRI (CD89) triggers different forms of neutrophil death depending on the inflammatory microenvironment

FcαRI (CD89), the human Fc receptor for IgA, is highly expressed on neutrophil granulocytes. In this study, we show that FcαRI induces different forms of neutrophil death, depending on the inflammatory microenvironment. The susceptibility of inflammatory neutrophils from sepsis or rheumatoid arthritis toward death induced by specific mAb, or soluble IgA at high concentrations, was enhanced. Although unstimulated cells experienced apoptosis following anti-FcαRI mAb stimulation, preactivation with cytokines or TLR agonists in vitro enhanced FcαRI-mediated death by additional recruitment of caspase-independent pathways, but this required PI3K class IA and MAPK signaling. Transmission electron microscopy of FcαRI-stimulated cells revealed cytoplasmic changes with vacuolization and mitochondrial swelling, nuclear condensation, and sustained plasma membrane. Coculture experiments with macrophages revealed anti-inflammatory effects of the partially caspase-independent death of primed cells following FcαRI engagement. Our data suggest that FcαRI has the ability to regulate neutrophil viability and to induce different forms of neutrophils depending on the inflammatory microenvironment and specific characteristics of the ligand-receptor interactions. Furthermore, these findings have potential implications for FcαRI-targeted strategies to treat neutrophil-associated inflammatory diseases.

Inhibition of ongoing allergic reactions using a novel anti-IgE DARPin-Fc fusion protein

Aggregation of the high-affinity IgE receptor (FcεRI) with the low-affinity IgG receptor (FcγRIIb) on basophils or mast cells has been shown to inhibit allergen-induced cell degranulation. Molecules cross-linking these two receptors might therefore be of interest for the treatment of allergic disorders. Here, we demonstrate the generation of a novel bispecific fusion protein efficiently aggregating FcεRI-bound IgE with FcγRIIb on the surface of basophils to prevent pro-inflammatory mediator release.

Determination of intrarenal resistance index (RI) in patients with multiple myeloma

Renal impairment is a common complication of multiple myeloma occurring in up to 50 % of patients at some stage in their disease. Due to occurrence of cast nephropathies we hypothesized circulatory dysregulation (vasoconstriction) in the kidneys with measurable elevation of the resistance index among these patients which would have a diagnostic impact.

Role of complement and Fc{gamma} receptors in the protective activity of the long pentraxin PTX3 against Aspergillus fumigatus

Pentraxin 3 (PTX3) is a soluble pattern recognition molecule playing a nonredundant role in resistance against Aspergillus fumigatus. The present study was designed to investigate the molecular pathways involved in the opsonic activity of PTX3. The PTX3 N-terminal domain was responsible for conidia recognition, but the full-length molecule was necessary for opsonic activity. The PTX3-dependent pathway of enhanced neutrophil phagocytic activity involved complement activation via the alternative pathway; Fc receptor (Fc R) IIA/CD32 recognition of PTX3-sensitized conidia and complement receptor 3 (CR3) activation; and CR3 and CD32 localization to the phagocytic cup. Gene targeted mice (ptx3, FcR common chain, C3, C1q) validated the in vivo relevance of the pathway. In particular, the protective activity of exogenous PTX3 against A fumigatus was abolished in FcR common chain-deficient mice. Thus, the opsonic and antifungal activity of PTX3 is at the crossroad between complement, complement receptor 3-, and Fc R-mediated recognition. Because short pentraxins (eg, C-reactive protein) interact with complement and Fc R, the present results may have general significance for the mode of action of these components of the humoral arm of innate immunity.

Individual IL-3 priming is crucial for consistent in vitro activation of donor basophils in patients with chronic urticaria

The in vivo autologous serum skin test (ASST) is the diagnostic gold standard to detect autoantibodies against FcεRI or IgE itself, as well as other autoreactive serum components, in patients with chronic spontaneous urticaria (CU). Coincubation of patient sera with donor basophils and measuring their degranulation in vitro could be a safe alternative but has shown inconsistent results.

Accelerated disassembly of IgE-receptor complexes by a disruptive macromolecular inhibitor

IgE antibodies bind the high-affinity IgE Fc receptor (FcεRI), found primarily on mast cells and basophils, and trigger inflammatory cascades of the allergic response. Inhibitors of IgE-FcεRI binding have been identified and an anti-IgE therapeutic antibody (omalizumab) is used to treat severe allergic asthma. However, preformed IgE-FcεRI complexes that prime cells before allergen exposure dissociate extremely slowly and cannot be disrupted by strictly competitive inhibitors. IgE-Fc conformational flexibility indicated that inhibition could be mediated by allosteric or other non-classical mechanisms. Here we demonstrate that an engineered protein inhibitor, DARPin E2_79 (refs 9, 10, 11), acts through a non-classical inhibition mechanism, not only blocking IgE-FcεRI interactions, but actively stimulating the dissociation of preformed ligand-receptor complexes. The structure of the E2_79-IgE-Fc(3-4) complex predicts the presence of two non-equivalent E2_79 sites in the asymmetric IgE-FcεRI complex, with site 1 distant from the receptor and site 2 exhibiting partial steric overlap. Although the structure is indicative of an allosteric inhibition mechanism, mutational studies and quantitative kinetic modelling indicate that E2_79 acts through a facilitated dissociation mechanism at site 2 alone. These results demonstrate that high-affinity IgE-FcεRI complexes can be actively dissociated to block the allergic response and suggest that protein-protein complexes may be more generally amenable to active disruption by macromolecular inhibitors.

A time-resolved fluorescence resonance energy transfer assay suitable for high-throughput screening for inhibitors of immunoglobulin E-receptor interactions

The interaction of immunoglobulin E (IgE) antibodies with the high-affinity receptor, FcεRI, plays a central role in initiating most allergic reactions. The IgE-receptor interaction has been targeted for treatment of allergic diseases, and many high-affinity macromolecular inhibitors have been identified. Small molecule inhibitors would offer significant advantages over current anti-IgE treatment, but no candidate compounds have been identified and fully validated. Here, we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for monitoring the IgE-receptor interaction. The TR-FRET assay measures an increase in fluorescence intensity as a donor lanthanide fluorophore is recruited into complexes of site-specific Alexa Fluor 488-labeled IgE-Fc and His-tagged FcεRIα proteins. The assay can readily monitor classic competitive inhibitors that bind either IgE-Fc or FcεRIα in equilibrium competition binding experiments. Furthermore, the TR-FRET assay can also be used to follow the kinetics of IgE-Fc-FcεRIα dissociation and identify inhibitory ligands that accelerate the dissociation of preformed complexes, as demonstrated for an engineered DARPin (designed ankyrin repeat protein) inhibitor. The TR-FRET assay is suitable for high-throughput screening (HTS), as shown by performing a pilot screen of the National Institutes of Health (NIH) Clinical Collection Library in a 384-well plate format.

An engineered disulfide bond reversibly traps the IgE-Fc3-4 in a closed, nonreceptor binding conformation

IgE antibodies interact with the high affinity IgE Fc receptor, FcεRI, and activate inflammatory pathways associated with the allergic response. The IgE-Fc region, comprising the C-terminal domains of the IgE heavy chain, binds FcεRI and can adopt different conformations ranging from a closed form incompatible with receptor binding to an open, receptor-bound state. A number of intermediate states are also observed in different IgE-Fc crystal forms. To further explore this apparent IgE-Fc conformational flexibility and to potentially trap a closed, inactive state, we generated a series of disulfide bond mutants. Here we describe the structure and biochemical properties of an IgE-Fc mutant that is trapped in the closed, non-receptor binding state via an engineered disulfide at residue 335 (Cys-335). Reduction of the disulfide at Cys-335 restores the ability of IgE-Fc to bind to its high affinity receptor, FcεRIα. The structure of the Cys-335 mutant shows that its conformation is within the range of previously observed, closed form IgE-Fc structures and that it retains the hydrophobic pocket found in the hinge region of the closed conformation. Locking the IgE-Fc into the closed state with the Cys-335 mutation does not affect binding of two other IgE-Fc ligands, omalizumab and DARPin E2_79, demonstrating selective blocking of the high affinity receptor binding.

The GPVI - Fc Fusion Protein Revacept Reduces Thrombus Formation and Improves Vascular Dysfunction in Atherosclerosis without Any Impact on Bleeding Times

Glycoprotein VI (GPVI) is a key platelet receptor which mediates plaque-induced platelet activation and consecutive atherothrombosis, but GPVI is also involved in platelet-mediated atheroprogression. Therefore, interference in GPVI-mediated platelet activation has the potential to combine short-term and long-term beneficial effects, specificity and safety especially regarding bleeding complications.