Klinik für Dermatologie, Venerologie und Allergologie Allergie-Centrum-Charité

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1 Klinik für Dermatologie, Venerologie und Allergologie Allergie-Centrum-Charité Allergy School on Allergy Diagnosis in & beyond the Skin am 25./ in Erlangen Session I -IgE-mediated/histamine mediated diseases: Principles of tests and their limitations Vortrag: "Basophil activation markers in anaphylaxis" Prof. Dr. med. Margitta Worm 2013 U N I V E R S I T Ä T S M E D I Z I N B E R L I N

2 Pathways of basophil activation and functional readouts. Kleine-Tebbe et al, Int Arch Allergy Immunol, 2006

3 Schematic representation of basophil activation caused by IgE receptor triggering. Crosslinking of high-affinity IgE receptors following allergen binding leads to the phosphorylation of Lyn and Syk tyrosine kinases which activate PI 3- kinase and phospholipase C (PLC). While PI 3-kinase may activate certain isoforms of phospholipase C, others may be activated by tyrosinekinases still needing to be identified in basophils. Following phospholipase Cactivation, IP 3 generation leads to a release of calcium stores into the cytosol which then facilitates a further rise in intracellular calcium caused by extracellular influx of the ion. IP 3 -mediated calcium mobilization is essential for the control of production and secretion of all main basophil mediator types. Less is known regarding the other product of phospholipase C activity, namely diacylglycerol and subsequent activation of protein kinase C (PKC), which exists in numerous isoforms having varied actions on mediator release. Calcium mobilization activates p38 MAPK, which plays a universally important role in governing the release of all mediator classes in basophils; however, ist exact interplay with calcineurin and ERK is still unclear. ERK is essential in the synthesis of LTC 4 in basophils, but has less effects on controlling other mediators. Basophil histamine and cytokine releases are extremely sensitive to the actions of calcineurin antagonists, suggesting a role for this enzyme in basophil signaling, but this is based solely on pharmacological evidence. For the sake of visual simplicity, arrows sometimes denote either a direct activation/modulation or indirect influence. Kleine-Tebbe et al, Int Arch Allergy Immunol, 2006

4 Variability and quantification of basophil responses. aige-/fc RI-mediated basophil functions demonstrate highly variable dose-response curves between different donors or within the same donor applying different stimuli (i.e. allergens). bbasophil reactivity (maximum of the basophil response) and basophil sensitivity, the (allergen) concentration inducing a distinct response, are used to analyze IgE-/Fc RI-mediated basophil responses. cboth variables, basophil reactivity (in this example C 1 B 1 A)and basophil sensitivity (C! B! A) are independent from each other. Kleine-Tebbe et al, Int Arch Allergy Immunol, 2006

5 Variables determining basophil responses. Arrows indicate directinfluence of one variable on another. Straight lines indicate independence ofneighboring variables from each other. Kleine-Tebbe et al, Int Arch Allergy Immunol, 2006

6 Basophil Fc RI receptor medians and ranges Kleine-Tebbe et al, Int Arch Allergy Immunol, 2006

7 In resting basophils CD63 is mainly anchored in the basophilic granule and is only weakly expressed on the surface membrane. On allergen-activated basophils, as a result of fusion between the granule and plasma membrane, CD63 is expressed with high density and mirrors mediator release. This up-regulated expression of basophilic CD63 can be detected by multicolour flow cytometry using specific anti-human IgE and anti-cd63 monoclonal antibodies. FITC, fluorescein isothiocyanate. Ebo et al, Clin Exp Allergy, 2004

8 Time-dependent upregulation of CD203c by recombinant rphl p 5 on basophils in a sensitized individual. Blood basophils were incubated with recombinant Phl p 5 (1 μg/ml) at 37 C for various time periods, as indicated, and then analyzed for expression of CD203c by flow cytometry. Hauswirth et al, J Allergy Clin Immunol, 2002

9 Dose-dependent effect of recombinant allergens on expression of CD203c in a sensitized individual. Whole blood was obtained from a patient allergic to Bet v 1 but not Bet v 2. Cells were incubated with serial dilutions of recombinant allergens or anti-ige at 37 C for 15 minutes, as indicated. Cells were then stained with CD203c mab and analyzed on a FACScan. Hauswirth et al, J Allergy Clin Immunol, 2002

10 Desensitization of basophils. Peripheral blood MNCs of an allergic patient were preincubated with allergens (rphl p 1, rphl p 2, rphl p 5, and rbet v 1, each at 0.1 μg/ml) in desensitizing PIPES buffer (D) or control medium for 45 minutes. After washing, MNCs were exposed to recombinant allergens (1 μg/ml each) in PIPES buffer containing Ca2+ for 15 minutes. After washing, cells were stained with the phycoerythrin-labeled CD203c mab 97A6. Hauswirth et al, J Allergy Clin Immunol, 2002

11 Gating procedure in CD203c-based BAT. Basophils gated in the lymphocyte region of the SSC/FSC pattern were selected as a HLA-DRneg/CD123pos population Sturm et al, Cytometry B Clin Cytom, 2010

12 Ex vivo basophil activation using native and roasted hazelnut (HN) extracts at different allergen concentrations (x-axis), in comparison with unstimulated cells and, as positive control, anti-ige- and buffer-stimulated cells. Worm et al, Clin Exp Allergy, 2009

13 In vivo basophil activation in hazelnut (HN)-allergic patients after native HN provocation in comparison with the control group (after intake of 10 g HN). Worm et al, Clin Exp Allergy, 2009

14 (a)cd203c expression in one patient before and after hazelnut (HN) provocation. (b) CD203c expression in one control individual before and after HN provocation. Worm et al, Clin Exp Allergy, 2009

15 Storage time-dependent decrease in anti-ige induced basophil activation. Blood samples from controls were stored for up to 48 h at 4 C before BAT was performed (anti-ige, 20 min, and 37C). Best responses were obtained when blood samples were processed immediately. A storage time from 24 to 48 h resulted in significantly reduced basophil activation. Data are expressed as means SEM, n ¼18; * P < 0.05 vs 0 h. baseline response (10%);--- threshold of 25% activated basophils. Sturm et al, Cytometry B Clin Cytom, 2010

16 Storage time-dependent decrease in basophil activation to bee and wasp venom. BAT was performed immediately after blood taking or after storage of 18 h at 4C with (A) bee (n ¼13) or (B) wasp venom (n ¼18) at the indicated concentrations. Data are expressed as means SEM, n ¼13 18; * P < 0.05 vs. 0 h. baseline response (10%); ---threshold of 25% activated basophils. Sturm et al, Cytometry B Clin Cytom, 2010

17 References 1. Hauswirth et al, Recombinant allergens promote expression of CD203c on basophils in sensitized individuals, J Allergy Clin Immunol, Ebo et al, In vitro allergy diagnosis: should we follow the flow?, Clin Exp Allergy, Kleine-Tebbe et al, Diagnostic Tests Based on Human Basophils: Potentials, Pitfalls and Perspectives, Int Arch Allergy Immunol, Worm et al, Impact of native, heat-processed and encapsulated hazelnuts on the allergic response in hazelnut-allergic patients, Clin Exp Allergy, Sturm et al, CD203c-Based Basophil Activation Test in Allergy Diagnosis: Characteristics and Differences to CD63 Upregulation, Cytometry B Clin Cytom, 2010