Chronic urticaria sera increase basophil CD203c expression

BACKGROUND: Approximately 40% of patients with chronic idiopathic urticaria have antibodies to the alpha subunit of the high-affinity IgE receptor. CD203c is a basophil activation marker known to be upregulated by cross-linking of the FcepsilonRIalpha receptor and may serve as a useful marker to identify these patients. OBJECTIVE: The primary objective was to assess the affect of sera from patients with chronic idiopathic urticaria on basophil CD203c expression. Secondary objectives were to correlate CD203c expression with basophil histamine release and size of the autologous serum skin test and to determine whether the mechanism is mediated by an IgG antibody. METHODS: Sera were obtained from patients with chronic idiopathic urticaria and positive autologous serum skin test or negative autologous serum skin test and normal controls. Sera were incubated with donor whole blood. Activated basophils from whole blood were identified by flow cytometry on the basis of the presence of CD203c on high-expressing IgE positive cells. RESULTS: Incubation of donor basophils with sera from patients with chronic idiopathic urticaria and positive autologous serum skin test demonstrated significant upregulation of CD203c. IgG depletion of representative sera from patients with chronic idiopathic urticaria resulted in significant decrease in CD203c expression on donor basophils. CD203c expression correlated with basophil histamine release and the size of the autologous serum skin test. CONCLUSION: Sera from patients with chronic idiopathic urticaria and positive autologous serum skin test significantly upregulate basophil CD203c and correlate with basophil histamine release. CLINICAL IMPLICATIONS: This article describes an activation marker on basophils whose expression is increased by sera from patients with chronic idiopathic urticaria.     

Regulation of allergic inflammation by the ectoenzyme E-NPP3 (CD203c) on basophils and mast cells

Adenosine 5′-triphosphate (ATP) is released from dying or damaged cells, as well as from activated cells. Once secreted, extracellular ATP induces several immune responses via P2X and P2Y receptors. Basophils and mast cells release ATP upon FcεRI-crosslinking, and ATP activates basophils and mast cells in an autocrine manner. Nucleotide-converting ectoenzymes, such as E-NTPD1, E-NTPD7, and E-NPP3, inhibit ATP-dependent immune responses by hydrolyzing ATP, thereby contributing to immune response regulation. E-NPP3 is a well-known activation marker for human basophils. E-NPP3’s physiologic function has recently been disclosed in mice. E-NPP3 is rapidly induced on basophils and mast cells after FcεRI-crosslinking and hydrolyzes extracellular ATP on cell surfaces to prevent ATP-dependent excess activation of basophils and mast cells. In the absence of E-NPP3, basophils and mast cells are overactivated and mice suffer from severe chronic allergic inflammation. Thus, the ATP-hydrolyzing ectoenzymes E-NPP3 has a nonnegligible role in the regulation of basophil- and mast cell-mediated allergic responses.     

Expression of CD203c and CD63 in human basophils: relationship to differential regulation of piecemeal and anaphylactic degranulation processes

Background Activation of human basophils results in the release of many different mediators and the expression of new cell surface proteins. The markers CD63 and CD203c have been used in recent years to assess basophil activation but there have been many studies that demonstrate that expression of these markers can be dissociated from histamine release. Objective To determine the signal transduction requirements for CD203c and CD63 expression. Methods The current study began by exploring the dependency of CD203c and CD63 expression on protein kinase C (PKC) using known selective inhibitors of PKC. Results Between 30 and 300 nm , Ro‐31‐8220 and bisindoylmaleimide II (Bis II) had no effect on formyl–met–leu–phe‐ or anti‐IgE‐induced CD63 or CD203c but enhanced IgE‐mediated expression of CD63 by an average of 15‐fold at concentrations >1 μm . These results led to the suggestion that these inhibitors altered the normal pathways of degranulation (by a non‐PKC dependent mechanism), shifting the normal presence of piecemeal degranulation to the process termed anaphylactic degranulation (AND). Morphological studies demonstrated that concentrations of Ro‐31‐8220 and Bis II>1 μm dramatically increased the presence of degranulation sacs, a morphological feature of AND. Conclusion It is proposed that CD63 expression results from only the AND form of histamine release. Cite this as: D. MacGlashan Jr, Clinical & Experimental Allergy, 2010 (40) 1365–1377.     

Inhibition of CD203c membrane up-regulation in human basophils by high dilutions of histamine: a controlled replication study

Objective  

Previous research suggests that human basophil activation may be inhibited by histamine even at extremely low doses (high dilutions). However, uncertainties about the nature of the phenomenon and its reproducibility mean that further, rigorously controlled studies are necessary.     

Cerivastatin and atorvastatin inhibit IL-3-dependent differentiation and IgE-mediated histamine release in human basophils and downmodulate expression of the basophil-activation antigen CD203c/E-NPP3

Recent data suggest that the statins, apart from their lipid-lowering activity, exhibit profound anti-inflammatory effects. Basophils are major proinflammatory effector cells in diverse pathologic reactions. We have examined the in vitro effects of five different statins on primary human basophils, their progenitors, and the basophil cell line KU-812. Preincubation of blood basophils with cerivastatin or atorvastatin (0.1-100 microM) for 24 h reduced their capacity to release histamine on immunoglobulin E (IgE)-dependent stimulation in a dose-dependent manner. These statins also inhibited IgE-dependent up-regulation of the basophil-activation antigen CD203c. Moreover, both statins suppressed interleukin-3-induced differentiation of basophils from their progenitors as well as (3)H-thymidine uptake in KU-812 cells. All inhibitory effects of cerivastatin and atorvastatin were reversed by mevalonic acid (200 microM). The other statins tested (lovastatin, simvastatin, pravastatin) did not show significant inhibitory effects on basophils. Together, these data identify cerivastatin and atorvastatin as novel inhibitors of growth and activation of human basophils.     

Comparison of basophil activation tests using CD63 or CD203c expression in patients with insect venom allergy

BACKGROUND: Flow cytometric basophil activation tests have been developed as cellular tests for in vitro diagnosis of IgE-mediated reactions. Different activation markers (CD63 or CD203c) with distinct ways of regulation have been used after stimulation with various allergens. OBJECTIVE: It was the aim of the present study to compare basophil activation tests by measuring both CD63 and CD203c upregulation in patients with insect venom allergy. MATERIALS AND METHODS: 43 patients with a history of insect venom anaphylaxis were examined. A careful allergy history was taken, and skin tests and determination of specific IgE-antibodies were performed. Basophil activation tests (BAT) using CD63 or CD203c expression were done after stimulation with different concentrations of bee and wasp venom extracts. 25 healthy subjects with negative history of insect venom allergy were studied as controls. RESULTS: The CD203c protocol showed a slightly higher sensitivity than the CD63 protocol (97% vs. 89%) with regard to patients' history. The magnitude of basophil response was higher with CD203c in comparison to CD63 for both insect venoms. Specificity was 100% for the CD63 protocol and 89% for the CD203c protocol with regard to controls with negative history and negative RAST. CONCLUSION: These results support the reliability of basophil activation tests using either CD63 or CD203c as cellular tests in the in vitro diagnosis of patients with bee or wasp venom allergy with a slightly higher sensitivity for the CD203c protocol.     

Depolarizing action of allergens on passive sensitized lymphocytes

An ability of rat thymus lymphocytes to respond to pollen allergens has been found. The lymphocytes were exposed with plasma of pollinosis patients. The subsequent addition of allergens specific for the patient caused the decrease of lymphocyte plasma membrane potential. The decrease was detected by use of potential-sensitive fluorescent probe 4-(p-dimethylaminostyryl)-1-methylpyridinium. This newly found effect can be employed for diagnostics of atopic states.     

Individual hymenoptera venom compounds induce upregulation of the basophil activation marker ectonucleotide pyrophosphatase/phosphodiesterase 3 (CD203c) in sensitized patients

BACKGROUND: Bee and wasp venom extracts contain potent allergens capable of inducing severe clinical reactions. To analyze immediate-type hypersensitivity to defined hymenoptera venom components, a recently developed in vitro test was applied that is based on the upregulation of CD203c expression on basophils. METHODS: CD203c expression on blood basophils of 9 healthy donors and 39 patients allergic to bee and/or wasp venom was analyzed by flow cytometry before and after activation with the purified bee venom allergens phospholipase A2 (Api m 1), hyaluronidase (Api m 2) and melittin (Api m 4), or the purified wasp venom allergens phospholipase A1 (Ves v 1), hyaluronidase (Ves v 2) and the recombinant antigen 5 (Ves v 5). Venom-induced CD203c upregulation on basophils was compared with skin tests and assessment of specific IgE. Basophils of nonresponders were preincubated with 10 ng/ml interleukin-3 (IL-3) prior to allergen stimulation. RESULTS: CD203c upregulation on basophils was induced by defined hymenoptera venom components in 35/39 patients with a diagnosed allergy to wasp and/or bee venom. Twenty-seven of the 34 tested patients with wasp allergy showed CD203c upregulation in response to Ves v 5, 26 of these patients also reacted with Ves v 2 and 17 with Ves v 1. Nine of 13 patients with bee allergy reacted with Api m 1, 13 individuals with Api m 2 and none of these patients with the minor allergen Api m 4. A diagnosed wasp allergy could also be confirmed in the prestimulated basophils (IL-3) of 2 nonresponder individuals who failed to upregulate CD203c in response to IgE receptor cross-linking prior to culture with IL-3. CONCLUSIONS: Flow-cytometric determination of CD203c upregulation on basophils activated by molecularly defined allergens is a powerful method to identify the precise allergen reactivity in sensitized individuals.     

Increased Level of Basophil CD203c Expression Predicts Severe Chronic Urticaria

Increased FcεR1α expression with upregulated CD203c expression on peripheral basophils is seen in patients with chronic urticaria (CU). However, there has been no published report on the association between CD203c expression level and clinical disease activity in CU patients. To investigate whether the increase of basophil activation is associated with the disease activity of CU, we measured basophil CD203c expression using a tricolor flow cytometric method in 82 CU patients and 21 normal controls. The relationship between the percentage of CD203c-expressing basophils and clinical parameters was analyzed. The mean basophil CD203c expression was significantly higher in CU patients than in healthy controls (57.5% vs 11.6%, P < 0.001). The basophil CD203c expression in severe CU patients was significantly higher than in non-severe CU (66.5% ± 23.3% vs 54.0% ± 23.3%, P = 0.033). Multiple logistic regression analysis indicated that both ≥ 72% basophil CD203c expression and urticaria activity score (UAS)≥ 13 were significant predictors of severe CU (P = 0.005 and P = 0.032, respectively). These findings suggest that the quantification of basophil activation with CD203c at baseline may be used as a potential predictor of severe CU requiring another treatment option beyond antihistamines.     

Marked improvement of the basophil activation test by detecting CD203c instead of CD63

BACKGROUND: The flow cytometric basophil activation test by detection of CD63 expression has been developed as an alternative method for in vitro diagnosis of IgE-mediated reactions to various allergens. Despite promising initial studies, the test remains disappointing in terms of sensitivity. CD203c has recently been demonstrated as a specific activation marker of basophils that is rapidly up-regulated after allergen challenge in sensitized patients. OBJECTIVE: The goal of the present study was to compare basophil activation tests by using either CD203c or CD63 in the diagnosis of immediate-type allergy to latex. METHODS: Twenty-seven patients (health care workers of our institution) who developed clinical features evocative of allergy after contact with latex were included and classified into two groups. Group 1 (n = 16) comprised true allergic patients who presented with typical signs of immediate allergic reaction associated with a positive skin test (prick test). Group 2 (n = 11) consisted of patients whose clinical history was not typical and had negative skin test. Twelve healthy subjects were also studied as controls. We compared the sensitivity of two triple-staining flow cytometric protocols measuring basophil activation after latex stimulation: CD45-IgE-CD63 and CD45-IgE-CD203c. RESULTS: The CD203c protocol showed a higher sensitivity than the CD63 protocol (75% vs. 50%). In comparison, latex-specific IgE sensitivity was found to be 69%. Furthermore, the magnitude of the basophil response was significantly higher with CD203c in comparison with CD63. Specificity was 100% for both protocols. CONCLUSION: Due to superior gating of basophils and a higher range of activation in response to allergen, the basophil activation test is markedly improved by use of CD203c instead of CD63.     

Comparison of two basophil activation markers CD63 and CD203c in the diagnosis of amoxicillin allergy

Background To confirm allergy to β‐lactam (BL), a basophil activation test in flow cytometry based on CD63 up‐regulation was described. CD203c is a more recent basophil activation marker and up to day there is no consensus about which marker is the more sensitive one. CD203c has not yet been evaluated in the diagnosis of BL allergy. Objective The aim of the study was to compare the reliability of CD203c to CD63 for the diagnosis of amoxicillin (AX) allergy, which is nowadays the most frequent BL allergy. Methods Twenty‐seven patients with an immediate positive skin test (ST) to AX, 20 had had anaphylaxis with AX and 7 had urticaria and/or angioedema, were compared with 14 controls with no allergy to BL and to six patients with delayed positive ST to AX. Results In the anaphylaxis group, AX induced up‐regulation of CD203c in the basophils of 12 patients out of 20 (60%) and of CD63 in four patients (20%) (P<0.02). Two patients out of seven with urticaria or angioedema had a positive result with CD203c and CD63. In patients who had anaphylaxis, ampicillin (AMP) induced CD203c up‐regulation in eight out of 12 (67%) patients tested, and CD63 up‐regulation in 4 out of 12 (33%) (all patients who had anaphylaxis could not be tested with AMP). False‐positive results were observed with CD203c as well as CD63, and for 10 patients indeed this was confirmed by a negative drug provocation test. The origin of conflicting results between CD63 and CD203c might be at least the targeting of basophils based on anti‐IgE labelling. Among IgE⁺ gated cells, by means of CD33, a marker of monocytes, a contamination up to 50% by monocytes was detected. In contrast to CD63, CD203c is an activation marker specific of basophils with a basal low‐level expression in resting basophils. Thus, IgE and CD203c double targeting of basophils avoids the contamination by monocytes. Conclusion CD203c seems to be a more sensitive activation marker of basophils than CD63 for the diagnosis of amoxicillin allergy.     

Simultaneous exposure of several allergens has an additive effect on multisensitized basophils

BACKGROUND: Patients immunoglobulin (Ig)E-sensitized to more than one allergen in their environment often have more symptoms than mono-sensitized individuals, which indicates that the allergens may have an additive effect. In order to study if such an effect could be detected on the inflammatory, cellular level, multisensitized basophils were challenged with various dose combinations of two relevant allergens. METHODS: Basophils from patients IgE-sensitized to timothy/cat, birch/cat, timothy/mite and cat/mite were challenged with serial dilutions of different combinations of the two allergens. The basophil response was measured as CD63 expression analysed by flow cytometry. RESULTS: The doses of each allergen in the pair had an additive effect resulting in a shift of the dose-response curve to higher CD63 percentages and higher CD-sens. CONCLUSIONS: If a patient has IgE antibodies and thus sensitized basophils to more than one allergen, to which he is simultaneously exposed, the additive effect should be considered. Even low concentrations of IgE antibodies could be of clinical relevance in such a situation.     

Flow cytometry for basophil activation markers: the measurement of CD203c up-regulation is as reliable as CD63 expression in the diagnosis of cat allergy

The flow cytometric basophil activation test (BAT), based on the detection of allergen-induced CD63 expression, has been proved effective in the diagnosis of various IgE-mediated allergies. However, there is not yet consensus about the suitability of CD203c expression as a specific basophil activation marker and its diagnostic reliability. The goal of the present study was to compare measurement of CD63 and CD203c expression using BAT in a model of cat allergy and to determine optimal experimental conditions for both markers. Heparinized whole blood samples from 20 cat allergic patients and 19 controls were incubated with Fel d1 (relevant allergen) or anti-FcepsilonRI (positive control) either in IL-3 or IL-3-free conditions. An optimal gating of basophils was achieved in triple staining protocols: anti-IgE PE/anti-CD45 PerCP/anti-CD63 FITC or anti-IgE FITC/anti-CD45 PerCP/anti-CD203c PE. We demonstrated that IL-3 significantly enhanced CD63-induced expression by basophils obtained from cat allergic patients in response to Fel d1. Sensitivity was found to be 100%. The CD203c protocol, when performed under IL-3-free conditions, also demonstrated 100% sensitivity. Only one of the control subjects was positive in both tests. In conclusion, using well-defined experimental conditions, the measurement of CD203c up-regulation on basophils in response to specific allergens is as reliable as CD63-BAT for the in vitro diagnosis of patients with IgE-mediated allergy.