Mast Cells Exhibiting Strong Cytoplasmic Staining for IgE and High Affinity IgE Receptor are Increased in IgG4-Related Disease

Immunoglobulin G4 (IgG4)-related disease is characterized by elevated serum IgG4 levels and increased numbers of IgG4-positive cells. However, its pathogenesis is not fully understood. We previously suggested that mast cells may play an important role in IgG4-related disease. In this study, we confirmed the characteristics of mast cells in IgG4-related lymphadenopathy by using immunohistochemistry and dual immunofluorescence. We analyzed 23 cases of IgG4-related lymphadenopathy and compared them with 23 cases of non-specific lymphoid hyperplasia. The majority of patients with IgG4-related lymphadenopathy had cervical lesions with involvement of other organs. Immunohistologically, mast cells with strong cytoplasmic staining for immunoglobulin E and high affinity immunoglobulin E receptor were significantly increased in IgG4-related lymphadenopathy as compared to those in non-specific lymphoid hyperplasia (mean: 3.83 ± 3.99 cells per high power field and 7.14 ± 8.21 cells per high power field, respectively; P = 0.007 and P = 0.011). In addition, dual immunofluorescence assay showed that immunoglobulin E and high affinity immunoglobulin E receptor staining exhibited a cytoplasmic granular pattern in IgG4-related lymphadenopathy, suggesting internalization of the antibodies and receptors. Our findings showed that mast cell activation might be involved in the pathogenesis of IgG4-related disease.

We previously reported a significant upregulation of IL-4, IL-10, IL-13, and transforming growth factor beta 1 in IgG4-related submandibular gland disease 9 . These findings in IgG4-related sialoadenitis were consistent with those of other previous reports 6,10,11 . In addition, we discerned that the infiltrating cells expressing Th2 and Treg cytokines were histomorphologically similar to mast cells and exhibited strong cytoplasmic staining for immunoglobulin E (IgE). Interestingly, although the number of c-kit-positive mast cells was not significantly increased in IgG4-related submandibular gland disease as compared to that in sialolithiasis and normal submandibular glands, the number of strongly cytoplasmic IgE-positive mast cells was significantly increased in IgG4-related submandibular gland disease. Based on these findings, we note mast cells exhibiting strong cytoplasmic staining for IgE as a key role in IgG4-RD. However, the underlying mechanisms and immunological significance of strong cytoplasmic IgE staining remain unclear.
In this study, we aimed to confirm whether mast cells exhibiting strong cytoplasmic staining for IgE were noted in IgG4-related lymphadenopathy cases and to identify the significance of the strong cytoplasmic staining.

Materials and Methods
Patients and samples. Tissue samples were obtained from 23 cases of IgG4-related lymphadenopathy.
Tissue samples from an equal number of cases (n = 23) of non-specific lymphoid hyperplasia were used as disease controls. All slides were jointly reviewed by K.N., M.T., and Y.S. Formalin-fixed paraffin-embedded specimens were used for immunohistochemistry and dual immunofluorescence. All tissue samples were obtained with the approval of the Institutional Review Board of Okayama University (Okayama, Japan). Informed consent for the use of their samples in research was obtained from all patients.
Histological examination and immunohistochemistry. The following methods were carried out in accordance with approved guidelines. All experimental protocols were approved by the Institutional Review Board at Okayama University.
Excisional biopsies were obtained from lymph node lesions in 23 patients with IgG4-related lymphadenopathy and an equal number of patients (n = 23) with non-specific lymphoid hyperplasia. The specimens were fixed in 10.0% formaldehyde and embedded in paraffin. Serial 4-µm-thick sections were cut from the paraffin-embedded tissue blocks and stained with hematoxylin and eosin. Sections were immunohistochemically stained using an automated Bond Max stainer (Leica Biosystems; Melbourne, Australia). The following primary antibodies were used: polyclonal IgG (dilution 1:20,000; Dako, Glostrup, Denmark), IgG4 (HP6025, dilution 1:10,000; The Binding Site, Birmingham, UK), KIT/CD117 (A4502, dilution 1:600; Dako), IgE (A094, dilution 1:500; Dako), and high affinity IgE receptor (FcεRI) (Ab54411, dilution 1:500; Abcam, Cambridge, UK). Following immunohistochemical staining, the number of IgG4-and IgG-positive cells were estimated in areas with the highest density of IgG4-positive cells. In accordance with the consensus statement on the pathological features of IgG4-RD 3 , three different high power fields (HPFs) (eyepiece: ×10, lens: ×40) were examined to calculate the average number of IgG4-positive cells per HPF and the IgG4-positive/IgG-positive cell ratio. IgE-and FcεRI-positive cells were counted in three different HPFs (eyepiece: ×10, lens: ×20) that were determined to have the highest density of positive cells. The average number of positive cells per HPF was calculated.
Dual immunofluorescence assays. For indirect dual immunofluorescence assays, paraffin sections were stained with the primary antibodies for IgE and FcεRI or FcεRI and c-kit. Fluorescein isothiocyanate-conjugated secondary antibodies (Alexa Fluor anti-mouse 555 and Alexa Fluor anti-rabbit 488; both from Life Technologies, Carlsbad, CA, USA) were used at a dilution of 1:400. The stained specimens were examined using a conventional immunofluorescence microscope (IX71; Olympus, Tokyo, Japan).

Statistical analysis.
Data are presented as the means and standard deviations. All statistical analyses were performed using the Mann-Whitney U test in the Statistical Package for the Social Sciences for Windows, software version 14.0 (SPSS Inc., Chicago, IL, USA). A P < 0.05 was considered statistically significant.

Results
Histological confirmation of IgG4-related lymphadenopathy. The clinicopathological characteristics of the patients with IgG4-related lymphadenopathy are summarized in Table 1. The cohort was comprised of 14 men and 9 women, with a median age of 61 (range, 45-82) years. Post-biopsy serum IgG or IgG4 levels were obtained for all patients except for Patient 23. On initial clinical examination, 14 patients presented with localized cervical lymphadenopathy. Seven patients had additional areas of lymphadenopathy (≥2) and 9 patients had extranodal lesions (e.g., in the salivary and lacrimal glands). All patients were followed up with regular imaging, laboratory assays, and clinical examinations. The median follow-up period was 42 (range, 2-154) months. During follow-up, 10 patients (43%) exhibited relapse in the residual lymph nodes or their disease had progressed to the development of other lymph nodes or extranodal lesions, including those of the submandibular and lacrimal glands. The average number of IgG-and IgG4-positive cells was 224.1 (range: 85.7-378) and 259.1 (range: 97.7-437.3) per HPF, respectively. In all cases, the IgG4-positive/IgG-positive cell ratios were >60.0%. The average numbers of IgE and FcεRI strongly cytoplasmic positive cells were 3.83 (range: 85.7-378) and 8.50 (range: 97.7-437.3) per HPF, respectively.
We confirmed that the tissue specimens from all 23 cases with IgG4-RD showed typical histological features of progressively transformed germinal center (PTGC)-type IgG4-related lymphadenopathy ( Fig. 1A-F).
Histological features of non-specific lymphoid hyperplasia. The specimens exhibited multiple lymphoid follicle formations with moderate lymphocytic infiltration of interfollicular areas. Compared to the histological features of IgG4-related lymphadenopathy, those of non-specific lymphoid hyperplasia showed no PTGC, and less infiltration of plasma cells and/or eosinophils. Dermatopathic and autoimmune disease-associated

IgE and FcεRI staining patterns in IgG4-related lymphadenopathy. A large number of IgE-and
FcεRI-positive cells were detected in the tissues of patients with IgG4-related lymphadenopathy and non-specific lymphoid hyperplasia ( Supplementary Figures 1 and 2). In the non-specific lymphoid hyperplasia specimens, most mast cells stained for IgE and FcεRI with a surface membrane pattern (Figs 2A and 3A). In contrast, in the IgG4-related lymphadenopathy specimens, some mast cells stained for IgE and FcεRI with a strongly cytoplasmic pattern (Figs 2B and 3B). There was a greater number of strongly cytoplasmic IgE-positive cells in the IgG4-related lymphadenopathy specimens than in the non-specific lymphoid hyperplasia specimens (mean: 3.83 ± 3.99 vs. 1.20 ± 1.79 cells per HPF, respectively; P = 0.007) (Fig. 2C). Strongly cytoplasmic FcεRI-positive cells were also detected more frequently in the IgG4-related lymphadenopathy specimens than in the non-specific lymphoid hyperplasia specimens (mean: 7.14 ± 8.21 vs. 2.13 ± 3.03 cells per HPF, respectively; P = 0.011) (Fig. 3C).
Dual immunofluorescence assay. In the IgG4-related lymphadenopathy specimens, IgE and FcεRI staining exhibited a cytoplasmic granular pattern (Fig. 4A,B). Intracytoplasmic granules stained positive for IgE (green) and FcεRI (red), some of which overlapped as yellow spots. In the non-specific lymphoid hyperplasia specimens, IgE and FcεRI stained the surface membrane. These cells were positive for c-kit.

Discussion
Although many previous studies regarding IgG4-RD have been reported in the last decade, the pathogenesis of the disease remains unclear. Recent studies have reported that IgG4-RD is a Th2-and Treg-dominant disease, and  Table 2. Clinical characteristics of IgG4-related lymphadenopathy and non-specific lymphoid hyperplasia. the activation of these cytokines may be responsible for this disease 5,6,9,10,[12][13][14][15] . However, studies investigating the mechanism underlying cytokine activation in IgG4-RD are few. In this study, we focused on mast cells exhibiting strong cytoplasmic staining for IgE. Comprehensive diagnostic criteria consisted of elevated serum IgG4 concentrations (>135 mg/dl), but not in all cases 3,16,17 . Some patients with PTGC-type IgG4-related lymphadenopathy have normal serum IgG4 levels when measurements are obtained after biopsy, at which time there are no residual primary lesions 18 . In our current data, serum IgG4 levels in 16 cases were <135 mg/dl. Furthermore, the recurrence ratio of IgG4-RD has been reported at 21.3% 17 . Some patients with PTGC-type IgG4-related lymphadenopathy have also experienced either persistence or relapse of residual lymph nodes 18 . In this study, 10 of 23 patients progressed to the development of other lymph nodes or extranodal lesions with elevated levels of serum IgG4.
Here, we confirmed that mast cells exhibiting strong cytoplasmic staining for IgE, were increased in IgG4-related lymphadenopathy. This is consistent with our previous report of submandibular gland disease 9 . Regarding the significance of strong cytoplasmic staining for IgE, we posit two hypotheses: overstaining and endocytosis.
IgG4-RD is frequently complicated by allergic conditions 19,20 . We previously reported that 52% of patients with IgG4-related lymphadenopathy also had allergic disease 18 . Our current study showed that 15 of 19 (78.9%) patients examined had allergic conditions. Furthermore, elevation of serum IgE levels, defined as IgE >250 IU/ ml, was observed in more than half of patients with IgG4-RD 17 , suggesting that strong cytoplasmic staining for IgE might indicate only overstaining. However, this study confirmed that mast cells exhibiting strong cytoplasmic staining for FcεRI also increased, as did staining for IgE.
Recent reports have shown that the activation of mast cells leads to internalization of IgE and FcεRI 21,22 . Accordingly, we added dual immunofluorescence assays, resulting in IgE and FcεRI staining exhibited as multiple cytoplasmic granules, some of which overlapped in IgG4-related lymphadenopathy. In contrast, staining in non-specific lymphoid hyperplasia was localized to the surface membrane. These results showed that the significance of strong cytoplasmic staining for IgE and FcεRI might reflect the internalization of IgE and FcεRI due to mast cell activation in IgG4-RD. Mast cells are activated by various biological substances (e.g., exogenous stimuli, endogenous peptides, chemokines, components of the complement system, and Fc receptors for IgE), leading to degranulation and the production of mediators such as cytokines and eicosanoids 23 . Some previous studies have suggested that response to antigen presentation might be involved in the pathogenesis of IgG4-RD 20,24-26 . Our current study suggests that some antigens might activate mast cells. However, additional assays are needed to elucidate the functional significance of IgE and FcεRI internalization. Although previous reports clarified the internalized FcεRI trafficking in early endosomes 23 , no published reports have investigated the trafficking completely.
Other Th subsets (T follicular helper cells or CD4+ cytotoxic T lymphocytes) and other immune cells (M2 macrophages) have recently been reported to participate in the pathogenesis of IgG4-RD 20,25,27-29 . Mattoo et al. 25 reported prominent clonal expansions of CD4+ SLAMF7+ cytotoxic T lymphocytes but not CD4+GATA3+ memory Th2 cells in patients with IgG4-RD. This strongly suggests that these cytotoxic T lymphocytes expanded in response to a specific causal antigen. Furukawa et al. 29 . suggested that M2 macrophages producing IL-33 are deeply involved in the activation of Th2 immune responses in IgG4-RD. Therefore, IgG4-RD might progress via the interconnected network of various Th subsets or immune cells.
In conclusion, we confirmed that mast cells exhibiting strong cytoplasmic staining for IgE and FcεRI were increased in IgG4-related lymphadenopathy, implying the activation of mast cells. Since mast cells "communicate" with various cells to produce a mediator, a better understanding of the role of mast cells could enable us to understand the pathogenesis of IgG4-RD.