Increases in IgE, Eosinophils, and Mast Cells Can be Used in Diagnosis and to Predict Relapse of IgG4-Related Disease

1IgG subclass 4–related disease (IgG4-RD) is a systemic fibroinflammatory condition, of which IgG4-related sclerosing cholangitis and autoimmune pancreatitis (AIP) are the biliary and pancreatic manifestations. An elevated serum IgG4 and abundance of IgG4-positive plasma cells infiltrating the involved organs is well described. Elevated serum IgE concentrations were first reported in a series of Japanese patients with AIP; retrospective studies suggest a prevalence of between 34% and 86%., Peripheral and tissue eosinophilia are traditionally seen in the context of atopic and allergic conditions, but have also been described in AIP. Furthermore, a clinical history of allergy can be ascertained in 40% to 80% of patients with AIP.,

2Strong links between IgG4 and IgE are well established. IgG4 responses are often associated with IgE-mediated allergy, but these responses are distinct. Both IgG4 and IgE are induced by Th2 cytokines interleukin (IL) 4 and/or IL13; however production of IgE antibodies often occurs well before IgG4 antibodies appear. It is also common to find IgG4 antibodies in the absence of IgE antibodies, a process called the “modified Th2 response.” An important regulatory component in the modified Th2 response is IL10, which promotes the switch to IgG4 and inhibits IgE production. IgG4 responses require frequent and/or high antigen exposure and are observed in situations associated with tolerance induction, such as during allergen immunotherapy. This can result in an increase of IgG4 (10–100 fold), along with a gradual decline in antiallergen IgE antibodies and symptoms of allergic rhinitis and asthma. Th2-cell-dominant immune responses are reportedly increased in the peripheral blood and tissue of IgG4-RD patients, and abundant regulatory cells producing IL10 and transforming growth factor-β infiltrate affected organs.,,

3In this paper, we set out to establish the prevalence of allergy and atopy, blood and tissue eosinophilia, serum total and allergen-specific IgE responses, and tissue IgE antibodies in a prospective UK cohort of IgG4-RD patients. We sought to determine the utility of serum IgE in differentiating patients with IgG4-related sclerosing cholangitis/AIP from disease mimics with an elevated serum IgG4, such as primary sclerosing cholangitis, and its relationship with corticosteroid use and disease relapse.

4For this study, 48 IgG4-RD patients, 42 disease control subjects with an elevated serum IgG4 (DC), and 51 healthy control subjects (HC) were recruited from the John Radcliffe Hospital, Oxford, United Kingdom, between March 2010 and March 2014, and followed-up for a median of 41 months (range, 3–73 months). Diagnostic criteria for each group are defined in the Supplementary Methods.

5Serum immunoglobulins were measured at diagnosis, during steroid treatment, and at disease relapse within the IgG4-RD cohort. Comparisons among IgG4-RD patients, DC, and HC were made at diagnosis before steroid initiation. Total serum IgG and IgG4 were measured by nephelometry (BNII analyser, Siemens, Oxford, UK). Total IgE was measured by the automated Immunocap method (Phadia 250, Milton Keynes, UK). Elevated serum IgG (≥16 g/L), IgG4 (≥1.4 g/L), and IgE (≥125 kIU/L) were defined by institution range.

6An IgE ImmunoCAP method was used to determine and quantify allergen-specific IgE antibodies. IgE-specific allergen testing was performed using a set of allergen mixes: grass pollen, mold, tree pollen, and nut (Phadia, Thermo Scientific, Uppsala, Sweden), as specified in the Supplementary Methods.

7Peripheral blood eosinophil count was measured at diagnosis. An elevated eosinophil count was defined as >500 cells/μL (count >0.5). Tissue eosinophil number was graded as none (0), occasional (<10), moderate (10–25), or marked (>25) on hematoxylin-eosin specimens by the reporting pathologist.

8Tissues were assessed for morphology and immunostained with IgG and IgG4 monoclonal antibody and IgE polyclonal antibody, as described in the Supplementary Methods. Double immunostaining for IgE and CD138 (plasma cells) or mast cell tryptase (mast cells) was performed.

9Comparisons among groups were analyzed with a 2-tailed Mann-Whitney test; correlations quantified as Spearman rank correlation coefficient. Statistics and receiver operating characteristic curve analysis were performed using Graphpad Prism (Graphpad Software, San Diego, CA) version 6.0. A P <.05 was considered significant.

10The serum IgG, IgG1, IgG4, and IgE levels at diagnosis were higher in IgG4-RD patients than in HC (P <.0001), as shown in Table 1 (Supplementary Figure 1A and B). There was no difference in the IgE/IgG4 ratio between the 2 groups (Supplementary Figure 1C). Elevated IgG4 concentrations were demonstrated in 81% (39 of 48), and elevated IgE in 54% (26 of 48) of the cases, both more prevalent compared with HC (P <.0001) (Table 1). There was a positive correlation between serum IgE and IgG4 (Spearman rank, 0.46; 95% confidence interval [CI], 0.18–0.66; P =.001) (Figure 1A), and serum IgE and IgG (Spearman rank, 0.32; 95% CI, 0.028–0.56; P =.028) (Supplementary Figure 2A). However, there was no difference between serum IgE levels in IgG4-RD patients with a normal and high IgG level (P =.178) (Supplementary Figure 2B). Serum IgE levels were increased in the high IgG4 compared with normal IgG4 group (P =.017) (Figure 1B). In the 9 IgG4-RD patients with a normal serum IgG4 at diagnosis, only 1 had an elevated IgE.

11Peripheral blood eosinophilia was present in 38% of IgG4-RD (median, 0.44; range, 0.0–5.05) versus 8.6% of HC (median, 0.16; range, 0.05–0.78) (P =.004) (Table 1). There was a positive correlation between eosinophil count and serum IgE (Spearman rank, 0.45; 95% CI, 0.18–0.65; P =.001) (Figure 1C and D) and serum IgG4 (Spearman rank, 0.34; 95% CI, 0.049–0.57; P =.019) (Supplementary Figure 2C). However, there was no difference between serum IgG4 levels in IgG4-RD patients with a normal and high eosinophil count (P =.298) (Supplementary Figure 2D).

12A history of allergy was identified in 63% of patients with IgG4-RD compared with 17% of HC (P <.001) (Table 1, Supplementary Figure 3A), and national UK statistics of 20% (http://www.allergyuk.org/allergy-statistics/allergy-statistics). There was a higher serum IgE in IgG4-RD patients with an allergic history than those without (P =.031) (Supplementary Figure 3B). An elevated serum IgE level was present in 60% of IgG4-RD patients with allergy. No patient had a history or evidence of parasitic infection.

13Atopy was defined by evidence of an IgE antibody response in addition to clinical symptoms. An allergen-specific IgE response was identified in 52% (25 of 48) of IgG4-RD patients and 40% (19 of 48) of IgG4-RD patients were defined as “atopic.” An elevated serum IgE level was present in 53% of IgG4-RD patients with atopy. In total, 62% (16 of 26) of IgG4-RD with an elevated total serum IgE had a positive allergen-specific IgE response and 41% (9 of 22) with a normal IgE had a positive IgE-allergen specific response (P =.25).

14To establish if there was a particular allergen prevalent in IgG4-RD and to investigate whether an IgE-specific response was more prevalent in those with an elevated total IgE, we tested the serum of 16 patients with elevated IgE and 17 patients with normal IgE levels to 4 broad allergen panels (grass, mold, tree, and nut mixes). Three-quarters (75%) of those with an elevated IgE (12 of 16) were positive to at least 1 of these panels, whereas 24% of those with a normal IgE (4 of 17) were positive to a single grass allergen panel only (P =.005) (Supplementary Table 1).

15Serum IgE levels were measured in IgG4-RD patients with an elevated IgG4, and DC who had other autoimmune, inflammatory, and infective diseases and an elevated IgG4, to investigate if IgE levels may help to discriminate the 2 groups. The groups were well matched in age (P =.21) and gender (P = 1.0). In those IgG4-RD patients with an elevated IgG4 (n = 39), serum IgE was higher (median, 323 kIU/L; range, 3.31–2024 kIU/L) than in the non-IgG4-RD DCs (n = 42) (median, 55.7 kIU/L; range, 5.11–4457 kIU/L) (P =.003) (Figure 2A). The ability of serum IgE to distinguish IgG4-RD from DC is shown on the receiver operating characteristic curve; area under the curve was 0.69 (P =.004; 95% CI, 0.57–0.81) (Figure 2B).

16At a serum IgE cutoff of 125 kIU/L, sensitivity was 67%, specificity was 64%, negative predictive value was 68%, and positive predictive value was 63%, with a likelihood ratio of 1.8 to distinguish IgG4-RD from non-IgG4-RD DCs with an elevated IgG4. At a serum IgE of 480 kIU/L (four times the upper limit of normal), the sensitivity fell to 36%, specificity increased to 86%, positive predictive value was 70%, and negative predictive value was 59%, with a likelihood ratio of 3.2.

17Of 48 IgG4-RD patients, 79% (38 of 48) received corticosteroid therapy, 15% (10 of 48) underwent surgical resection, and 6.2% (3 of 48) had conservative management because of severe diabetes (1), end-stage cirrhosis (1), and patient preference (1). In those who received corticosteroid therapy with available serum samples (n = 15), IgE levels were lower compared with pretreatment levels by 12 weeks (P <.001) (Supplementary Figure 4A). Over time, serum IgE levels plateaued or increased, corresponding to corticosteroid discontinuation and clinical remission (Supplementary Figure 4B).

18Of 48 IgG4-RD patients, 43 had sufficient follow-up (>6 months) to determine evidence of relapse. At least 1 episode of biochemical and/or radiologic relapse was recorded in 54% (23 of 43). Serum IgE at diagnosis was higher in those IgG4-RD patients who relapsed than those who did not (P =.001) (Figure 3A). Receiver operating characteristic curve analysis yielded an area under the curve of 0.76 (P =.005; 95% CI, 0.62–0.91). Using an IgE cutoff of 380 kIU/L, the sensitivity was 64%, specificity was 88%, with a likelihood ratio of 5.4, to predict relapse (Figure 3B). An IgE of >125 kIU/L at diagnosis did not predict relapse.

19Histologic specimens were available from 77% (37 of 48) of IgG4-RD patients for morphologic review and immunostaining; 15 resection and 32 biopsies. Tissue eosinophils were present in 86% (32 of 37) of specimens. Eosinophil infiltration was graded as “moderate” (10–25 eosinophils/high power field [HPF]) or “marked” (>25 eosinophils/HPF) in 73% (27 of 37) of specimens.

20Tissue IgG4 immunohistochemistry was performed in all 37 IgG4-RD specimens; the IgG4-positive plasma cell count >10/HPF (range, 5–130/HPF) in 95% (35 of 37). The 2 patients with biopsy IgG4 counts <10/HPF had resection specimens with morphologic characteristics and ratios “highly suggestive” of IgG4-RD.

21Tissue IgE immunohistochemistry was performed in 8 IgG4-RD and 6 DC specimens (Supplementary Table 2). The IgE-positive cell count in IgG4-RD (median, 10 cells/HPF; range, 1–30 cells/HPF) was higher than DC (median, 4 cells/HPF; range, 0–10 cells/HPF) (P =.15) tissues. The IgE-positive cell count was >10/HPF in 50% of samples stained (4 of 8). In IgG4-RD tissues, IgE-positive cells were localized to the mantle zone of the germinal center, within lymphoid aggregates and also scattered throughout the inflammatory infiltrate (Figure 4A). The IgE-positive cell staining was both nuclear and surface. Mast cells were also scattered throughout the inflammatory infiltrate (Figure 4B). IgE-positive cells and mast cells had a similar distribution, and costaining demonstrated evidence of IgE-positive mast cells (mast cell cytoplasmic staining and IgE peripheral staining) in IgG4-RD tissue (Figure 4C). IgE-positive cells stained separately to CD20-positive B cells (Figure 4D) and CD138-positive plasma cells (Figure 4E). This suggests IgE-positive cells represent IgE attachment to mast cells, although we cannot exclude the possibility that a proportion could be IgE-producing B cells.

22In 3 IgG4-RD patients with the highest mean count of IgE-positive mast cells, a retrospective serum mast cell tryptase was performed (presteroid samples), which were within the normal range.

23Red flags, incorporating these findings, are shown in Figure 5.

24In the original landmark study of elevated serum IgG4 in patients with AIP in Japan, there was no difference reported in IgE levels between AIP and HC. Subsequent retrospective studies reported an elevated IgE in 34%–86% of patients with AIP., In our prospective study of IgG4-RD, an elevated IgE was found in 57% at diagnosis, and a positive correlation was shown between IgE and both IgG4 and eosinophils count. We report a frequent clinical history of allergy (63%) and atopy (40%) in IgG4-RD, supported by retrospective data in Japanese patients with AIP, but not by other groups.,, Peripheral eosinophilia and elevated IgE have also been described in subsets of IgG4-RD patients without atopy. The main discrepancy lies in the definition of atopy; symptoms plus an IgE-specific protein response, which has not been characterized in other studies. We also show a higher IgE level in those individuals with allergy/atopy, supported by studies showing IgG4-RD patients with atopy express up-regulated Th2 cytokines in peripheral blood., We have also reported polyclonal elevations of IgG4 to multiple food allergens in IgG4-RD patients.

25Our results support the utility of serum IgE in diagnosis of IgG4-RD, with a level of >480 KU/L differentiating IgG4-RD from DC with an elevated serum IgG4 (specificity 86%, sensitivity 36%). This 2-dimensional diagnostic approach (ie, IgE combined with IgG4) was supported by the observation that patients with a normal serum IgG4 did not have an elevated IgE. There were no DCs with malignancy in this dataset; however, serum IgE levels may be used to distinguish organ-specific IgG4-RD from malignant lesions, potentially minimizing unnecessary surgical intervention. This approach requires further validation. An elevated IgE at diagnosis >380 KU/L was also a marker of disease relapse (specificity 88%, sensitivity 64%), defining a subgroup requiring careful follow-up. This is supported by findings in a recent retrospective cohort study of predictors of disease relapse in IgG4-RD after rituximab therapy; in 21 (37%) patients experiencing disease relapse, baseline serum IgG4, IgE, and circulating eosinophils predicted relapse.

26Interestingly, IgE levels decreased during the first 12 weeks of corticosteroid therapy, with a parallel fall in disease activity (and IgG4 levels). This finding is in line with observations in a retrospective cohort of AIP. Furthermore, the anti-CD20 B cell depletion therapy rituximab, used in refractory disease, results in a decrease in serum IgG4 and IgE with regression of active disease. However, IgE levels do not fall with steroid treatment in atopic dermatitis and do not reflect disease activity in asthma or allergic rhinitis. This complex interplay between allergic disease and IgE levels indicates that a correlation between IgE and response to treatment does not necessarily point to a pathogenic role for IgE in IgG4-RD.

27The presence of tissue IgE-positive mast cells in affected organs represents a novel finding, and suggests a possible role of an IgE-mediated response. Mast cells are involved in a variety of immune responses including chronic inflammation and autoimmune disease. IgE is a key stimulator of mast cells via binding to the high-affinity IgE receptor (FcεRI). Mast cells secrete mediators including Th2 and regulatory cytokines in response to allergens binding to specific cell-bound IgE. Nonspecific polyclonal IgE can also induce cytokine secretion, independent of antigen. Furthermore, chronic elevation of IgE induces upregulation of FcεRI on mast cells, resulting in inhibition of mast cell apoptosis and promotion of cytokine production. Indeed, this may explain the predominantly surface IgE-positive mast cell staining seen in our tissue specimens, caused by high levels of membrane-associated IgE. Mast cells might also contribute to fibrosis, supported by our observation of IgE-positive mast cell infiltration in an IgG4-related fibrosclerotic mesenteric mass (Supplementary Table 2).

28The etiology of the elevated IgG4 and IgE in the blood and tissue of IgG4-RD patients remains unexplained. Both IgG4 and IgE require Th2 cytokines, IL4 and IL13, for induction. By contrast, IL10 promotes IgG4-switched cells, but downregulates IgE. Although we have shown that tissue IgE-positive cells represent IgE attachment to mast cells, a proportion could also be IgE-producing B cells. IgE-producing memory B cells and plasma cells may arise directly through a germinal center IgE-intermediate cell, but also indirectly from class switching of IgG4 or IgG1 B cells. Both situations could lead to elevated IgE and IgG4 in IgG4-RD, the former by sharing of Th2 conditions and the latter in a higher frequency switch from IgG4 cells. Of note, IgG4 B cells seem to have increased capacity to capture IgE molecules, by upregulation of the FcεRII on their surface, suggesting a functional link between IgE and IgG4 B cell responses. Importantly, dysfunction of immune cells, such as Th2 and T regulatory cells, and cytokines IL4, IL13, and IL10, may pave the way for increasing serum IgE levels and IgE-positive cells in the tissues of patients with IgG4-RD.

29The discovery of IgE, eosinophilia, and mast cell infiltration in IgG4-RD highlights novel therapeutic options. The prostaglandin D2 receptor (CRTh2), important in allergic inflammation, is expressed on Th2 cells; innate cells, such as eosinophils; and responds to mast cell–derived factors., The frequency of CRTh2 cells was increased in IgG4-siloadenitis, with a nonsignificant correlation with IgE and eosinophils. We also report upregulation of PGD2 and CRTh2 in gene expression analysis of patients with IgG4-related sclerosing cholangitis/AIP. Blockade of CRTh2 can reduce allergic inflammation in rodent models of antigen-induced airway inflammation, allergic rhinitis, and atopic dermatitis, and may be effective in a subgroup of IgG4-RD patients with atopy.

30In summary, this prospective study provides new evidence for the relationship of serum and tissue IgE antibodies in patients with IgG4-RD. Firstly, it supports the measurement of serum IgE levels at diagnosis, to help differentiate IgG4-RD from non-IgG4-RD conditions, and to determine the risk of subsequent disease relapse in those with IgG4-RD. Secondly, it implicates an IgE-mediated allergic response in a subgroup of patients, supported by a history of allergy and atopy, peripheral and tissue eosinophilia and the presence of IgE+ mast cells in affected tissues, which enables novel therapeutic avenues to be explored.