Effector Cells and Mechanisms in Chronic Spontaneous Urticaria

Borzova, Elena (2014) Effector Cells and Mechanisms in Chronic Spontaneous Urticaria. Doctoral thesis, University of East Anglia.

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Abstract

Background: Chronic spontaneous urticaria (CSU) is characterised by weals, angioedema, or both, which occur for six weeks or more. Itchy, red and raised weals in CSU are thought to occur as a result of skin mast cell activation, local vasodilatation and increased vascular permeability which are the cardinal features of the disease. Serum histamine-releasing activity and abnormal basophil function were implicated in the pathophysiology of CSU. We hypothesized that severe and/or persistent CSU may be associated with serum histamine releasing activity (HRA), abnormal basophil releasability, numbers and phenotype. Furthermore, serum HRA in CSU was hypothesized to be associated with higher local concentrations of pro-inflammatory mediators (histamine, tryptase) and IL-6 in the skin, local histamine release and, possibly, neutrophil infiltration in the dermis. To test this hypothesis, we carried out cutaneous microdialysis of autologous serum skin test (ASST) response, a prospective study of basophil-relate biomarkers in CSU and a retrospective histological study in CSU and urticarial vasculitis (UV).
In cutaneous microdialysis study, 14 CSU patients and 13 healthy subjects were evaluated to determine the baseline levels of histamine, tryptase and cytokines and their changes in response to skin testing with PBS (pH = 7.4, 20μl), autologous serum (20μl) and codeine (0.3mM, 20μl). We demonstrated a slow low-grade local histamine release after intradermal injection of autologous serum in two HRA+ CSU patients. There was elevated dermal histamine (p=0.0193) but normal tryptase (p=0.1437) and IL-6 (p=0.1298) concentrations in CSU compared to healthy controls. Dermal histamine concentrations were correlated to clinical scores in CSU (r=0.602, p<0.05).
The prospective observational study was carried out in 22 CSU patients at three time points over 6 months to elucidate the relationship between the biomarkers to disease severity and disease persistence. Laboratory assessments included serum-induced basophil HRA on healthy donor basophils, anti-IgE-induced basophil histamine release (BHR) from CSU patients, basophil flow cytometry. Baseline UAS7 correlated with serum HRA (r=0.58, p=0.0045), and anti-IgE-induced BHR (r=0.40, p=0.0666).HRA+CSU patients (n=8) had a more severe disease than HRA- CSU patients (n=14) (p=0.0152). Based on the ROC analysis for UAS7 at baseline, a cut-off value of 19 predicted persistence of CSU in our patient population with 63.16% accuracy (sensitivity of 60% and specificity of 66.67%). These results should, however, be interpreted with caution in view of a small sample size and the selected patient population from the secondary care dermatological setting. There was a persistent (n=3) versus transient (n=3) increase in serum HRA in CSU patients over time. Flow cytometric enumeration in CSU varied depending on choice of gating strategy for peripheral blood basophils (CCR3+CD123+ vs CCR3+CD63+ (p=0.0001), CD63+CD203c+ vs CCR3+CD123+ (p=0.0003), CD63+CD203c+ vs CCR3+CD63 (p=0.0001)). We then examined basophil variation using ImageStream® in healthy subjects. ImageStream® studies confirmed the difference in basophil percentages detected by CD63+CD203c+ and CCR3+CD63+ gating strategies (0.02% vs 0.4% of basophils) in peripheral blood from a healthy donor following Ficoll-Paque density gradient centrifugation. In the CD203c+ OR CD63+ Boolean gate, we identified a basophil subpopulation with surface alterations that comprised 17.7% cells in this gate in peripheral blood sample from a healthy donor. All basophils with surface alterations were CD63+cells, 93.75% of which were CCR3+cells and 0.78% of which were CD203c+cells in this healthy donor.
In the retrospective study, we compared clinical and histological findings in CSU (n=33) and UV (n=43) patients for the presence of skin autoreactivity or serum HRA and eosinophils/neutrophil numbers per high power field (HPF). There were higher numbers of neutrophils/HPF in UV than CSU patients by haematoxylin and eosin (H&E) staining (p=0.0002) and immunohistochemical detection of myeloperoxidase (p=0.0001). Neutrophilic urticaria (more than 25 extravasated neutrophils per five HPF) was noted in 63.6% of CSU patients including two CSU patients with serum HRA.
Conclusions: In CSU, disease severity is associated with higher dermal histamine concentrations, serum HRA and abnormal basophil releasability. In ASST+ patients, an intradermal injection of autologous serum resulted in local in vivo histamine release suggesting that the ASST response is a useful experimental model of spontaneous wealing in CSU. Whether in vivo local histamine release explains the ASST response in CSU patients needs to be established in future studies. Baseline UAS7 is associated with serum HRA and anti-IgE-induced BHR and may predict disease persistence in CSU patients. Basophil phenotypic variation was demonstrated by different gating strategies which may reflect in vivo basophil activation in CSU. Biological and technical factors may contribute to basophil variation in imaging flow cytometry. In the lesional skin biopsies, neutrophil counts/HPF, using H&E staining or immunohistochemistry, can be useful for the diagnosis of neutrophilic urticaria and to differentiate between CSU and UV.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User:	Nicola Veasy
Date Deposited:	09 Mar 2015 14:19
Last Modified:	09 Mar 2015 14:19
URI:	https://ueaeprints.uea.ac.uk/id/eprint/52580
DOI:

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