Human eosinophils express the high affinity IgE receptor, FcεRI, in bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune blistering disease mediated by autoantibodies targeting BP180 (type XVII collagen). Patient sera and tissues typically have IgG and IgE autoantibodies and elevated eosinophil numbers. Although the pathogenicity of the IgE autoantibodies is established in BP, their contribution to the disease process is not well understood. Our aims were two-fold: 1) To establish the clinical relationships between total and BP180-specific IgE, eosinophilia and other markers of disease activity; and 2) To determine if eosinophils from BP patients express the high affinity IgE receptor, FcεRI, as a potential mechanism of action for IgE in BP. Our analysis of 48 untreated BP patients revealed a correlation between BP180 IgG and both BP180 IgE and peripheral eosinophil count. Additionally, we established a correlation between total IgE concentration and both BP180 IgE levels and eosinophil count. When only sera from patients (n = 16) with total IgE ≥ 400 IU/ml were analyzed, BP180 IgG levels correlated with disease severity, BP230 IgG, total circulating IgE and BP180 IgE. Finally, peripheral eosinophil count correlated more strongly with levels of BP180 IgE then with BP180 IgG. Next, eosinophil FcεRI expression was investigated in the blood and skin using several methods. Peripheral eosinophils from BP patients expressed mRNA for all three chains (α, β and γ) of the FcεRI. Surface expression of the FcεRIα was confirmed on both peripheral and tissue eosinophils from most BP patients by immunostaining. Furthermore, using a proximity ligation assay, interaction of the α- and β-chains of the FcεRI was observed in some biopsy specimens, suggesting tissue expression of the trimeric receptor form in some patients. These studies provide clinical support for the relevance of IgE in BP disease and provide one mechanism of action of these antibodies, via binding to the FcεRI on eosinophils

Patient characteristics and mRNA expression of FcεRI α, β, and γ receptor chains^a.

a<p>mRNA expression was evaluated by RT-PCR after immunomagnetic purification of eosinophils.</p>b<p>reported as IU/ml, normal range ≤100.</p>c<p>expressed as Index Units, positive test ≥19.</p>d<p>reported as BP index/BPDAI as described in the Methods.</p>e<p>Not done.</p><p>Patient characteristics and mRNA expression of FcεRI α, β, and γ receptor chains^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107725#nt110" target="_blank">a</a>}.</p

Eosinophils from active BP patients degranulate in response to BP180 protein.

<p>For degranulation, peripheral blood was incubated with 10 µg/ml NC16A or GST control protein in duplicate and EDN release was measured by ELISA. Mean GST values (background) were subtracted from NC16A and results are expressed as percent maximal (100 nM ionomycin) release. Each point represents the mean of replicate samples from the same patients. The number of patients per group is indicated. A Kruskal-Wallis test was performed, * = p≤0.05.</p

Disease activity and autoantibody profiles in patients with BP.

<p>Subjects (n = 48) were enrolled before receiving any immunosuppressive treatment for BP. Since patients were enrolled over several years, disease activity was scored with a BP Index (A) or the more comprehensive Bullous Disease Area Index (BPDAI) criteria (B). A strong correlation (Spearman’s r = 0.7193, p<0.0001) was observed between these two scoring systems (C). Sera were collected from untreated BP patients (BP sera; BPS), patients evaluated for other autoimmune skin diseases (other autoimmune sera; OAS), or age- and gender-matched controls (normal human sera; NHS) and evaluated for BP180 IgG, BP230 IgG, total IgE and BP180 IgE by ELISA. Each point represents the average of duplicate samples from an individual patient with the N per group indicated. Mann-Whitney U-test, *p≤0.05, **p≤0.01, ***p≤0.001.</p

Correlation^a between antibody levels, eosinophil counts, and disease severity in untreated BP patients.

a<p>determined using Spearman’s rank correlation coefficient (r) and p = *<0.05, **<0.01, ***0.001.</p>b<p>reported as IU/ml, normal range ≤100.</p>c<p>expressed as Index Units, positive test ≥19.</p>d<p>reported as BP index/BPDAI as described in the Methods.</p><p>Correlation^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107725#nt101" target="_blank">a</a>} between antibody levels, eosinophil counts, and disease severity in untreated BP patients.</p

Samples evaluated for FcεRI expression using the proximity ligation assay.

a<p>eosinophils were enriched from peripheral blood using density centrifugation and adhered to glass slides.</p>b<p>expressed as IU of IgE, normal range ≤100 IU.</p>c<p>ELISA Index Value, normal range ≤19.</p>d<p>reported as BP index/BPDAI as described in the Methods.</p>e<p>not done.</p><p>Samples evaluated for FcεRI expression using the proximity ligation assay.</p

Surface expression of FcεRI on BP eosinophils.

<p>Interaction of FcεRIα with IgE or FcεRIβ was evaluated using the proximity ligation assay on non-permeabilized preparations of circulating granulocytes or skin cryosections from BP patients or controls. Eosinophils were identified by their unique nuclear morphology (bi-lobed nucleus stained with DAPI) using high resolution confocal microscopy. Interaction of FcεRIα/IgE on peripheral blood and tissue eosinophils from BP patient (A, B) or controls (C, D). Insets are enlarged to show nuclear morphology. Scale bar = 25 uM. Interaction of FcεRIα/FcεRIβ on eosinophils in lesional biopsies (E–H). Panel H is an image of the same BP sample depicted in panel G, captured at higher magnification for resolution of nuclear morphology. Scale bar = 50 uM.</p

Evaluation of IgE receptor expression on circulating eosinophils from BP patients.

<p>Peripheral granulocytes were stained with fluorescently tagged antibodies specific for human CD203c, CD49d, CD16, and FcεRI-α, FcεRIβ, CD23 or IgE for flow cytometric analysis. Eosinophils were identified by gating on the CD16⁻/CD49d⁺/CD203c⁻ population. Eosinophils from healthy controls (A), active BP patients (B), or basophils (CD16⁻/CD49d⁺/CD203c⁺) from active BP patients (C) were evaluated. The degree of specific staining is indicated by the open histogram compared to appropriate isotype control shown by the shaded histogram. Staining is representative of 10 BP patients and 11 age- and gender-matched controls.</p

A qualitative study of alcohol, health and identities among UK adults in later life

Increasing alcohol consumption among older individuals is a public health concern. Lay understandings of health risks and stigma around alcohol problems may explain why public health messages have not reduced rates of heavy drinking in this sector. A qualitative study aimed to elucidate older people's reasoning about drinking in later life and how this interacted with health concerns, in order to inform future, targeted, prevention in this group. In 2010 a diverse sample of older adults in North East England (ages 50–95) participated in interviews (n = 24, 12 male, 12 female) and three focus groups (participants n = 27, 6 male, 21 female). Data were analysed using grounded theory and discursive psychology methods. When talking about alcohol use older people oriented strongly towards opposed identities of normal or problematic drinker, defined by propriety rather than health considerations. Each of these identities could be applied in older people's accounts of either moderate or heavy drinking. Older adults portrayed drinking less alcohol as an appropriate response if one experienced impaired health. However continued heavy drinking was also presented as normal behaviour for someone experiencing relative wellbeing in later life, or if ill health was construed as unrelated to alcohol consumption. Older people displayed scepticism about health advice on alcohol when avoiding stigmatised identity as a drinker. Drinking patterns did not appear to be strongly defined by gender, although some gendered expectations of drinking were described. Identities offer a useful theoretical concept to explain the rises in heavy drinking among older populations, and can inform preventive approaches to tackle this. Interventions should engage and foster positive identities to sustain healthier drinking and encourage at the community level the identification of heavy drinking as neither healthy nor synonymous with dependence. Future research should test and assess such approaches