An In Vitro Reporter Cell System for Analysis of Functional Ly49 Receptor Binding of Cognate Ligands in Cis and Tran

Activation of natural killer (NK) cells requires the integration of stimulatory and inhibitory signals mediated in part by members of the allelic Ly49 receptor family. Inhibitory Ly49 receptors bind cognate MHC class I ligands in trans (ITIM activation) and in cis (no ITIM activation). We have developed an in vitro reporter cell system for analysis of the functional interaction between the activating Ly49H receptor (C57BL/6 strain, B6) and its murine cytomegalovirus (MCMV)-encoded ligand, m157. Since m157 also binds inhibitory Ly49 receptors, including Ly49I from 129 mice, we exploited our reporter cell system to determine: 1) whether Ly49HB6 and/or Ly49I129 bind the GPI-linked m157 ligand in cis, and 2) whether the induction of beta-galactosidase (b-gal) reporter activity of Ly49H-expressing HD12 cells could be inhibited or attenuated by the co-expression of Ly49I129 (HD12-I129 cells) binding to the same m157 ligand. We found that Ly49HB6, but not Ly49I129, binds m157 in cis, as measured by flow cytometry and by activation of Ly49H reporter (HD12) cells. When Ly49I129 is co-expressed in cis with Ly49H and m157, partial m157 staining is restored, possibly reflecting trogocytosis of m157 by Ly49I129. When Ly49H is co-expressed with Ly49I129 and m157, the mean fluorescence intensity of m157 is slightly reduced and correlates with a minor reduction in HD12 activation (in trans). We also show that co-expression of Ly49HB6 and Ly49I129 on HD12-I129 cells results in lower b-gal induction following co-incubation with m157-expressing stimulator cells compared with HD12 cells (expressing only Ly49H). These findings represent a novel demonstration of cis ligand binding for an activating Ly49 receptor, and also demonstrate the utility of this reporter cell system for analysis of other relevant inhibitory and activating Ly49 receptor interactions (e.g. Ly49G2 and H-2Dk)

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

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

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

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

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