Abstract
Graves’ ophthalmopathy (GO) with elevated IgG4 levels has different characteristics from patients with GO who do not have elevated IgG4 levels, but the study findings are contradictory. The goal of this study was first to investigate the relationship between IgG4/IgG and IgG4 levels and the occurrence of GO and then to investigate the clinical and laboratory characteristics of patients with GO who had elevated IgG4 levels. This study control group consisted of 57 Graves’ disease(GD)patients with no complicated ocular disease and a median followup of 54.41 months, while the experimental group included 171 GO patients. The clinical and laboratory characteristics of the GD and GO groups were compared. Using an IgG4/IgG cut-off value of 8%, the GO patients were divided into two groups: high IgG4/IgG and low IgG4/IgG, and the differences in clinical and laboratory characteristics between these two groups, as well as between the GO patients before and after 3 months of treatment, were analyzed. The study results show that serum IgG4/IgG levels were independently correlated with the occurrence of GO, but not significantly correlated with GO activity. Patients with high IgG4/IgG levels of GO exhibit distinct characteristics, which may be associated with an unstable balance of lymphocyte subsets.
Keywords: Graves’ ophthalmopathy, Graves’ disease, IgG4/IgG, Clinical features
Subject terms: Endocrine system and metabolic diseases, Immunology
Introduction
IgG4-related disease (IgG4-RD) is an immune-mediated fibroinflammatory condition that can affect many different systems in the body. One of its characteristics is an increase in serum IgG4 levels, and the thyroid is also one of the affected organs1,2. Studies have shown that a small percentage of patients with thyroid disease exhibit characteristics similar to IgG4-RD, so it is referred to as thyroid disease with elevated IgG43. In healthy subjects, IgG4 is the least abundant IgG subclass, accounting for only 3–6% of total IgG4. GO is the most common extra-thyroid manifestation of GD, with a prevalence rate ranging from 25 to 50% depending on race. Although most patients have mild symptoms, 20–30% can develop moderate to severe symptoms, and 3–5% may lose their vision5,6. It has been demonstrated that GD patients with elevated IgG4 levels are more likely to develop GO, which is also linked to GO activity7–9. While also have studies shown that GD patients with elevated IgG4 levels are unrelated to the occurrence of GO, as well as the activity and severity of GO10. Whether IgG4 levels are related to the occurrence of eye complications in GD patients or whether GO patients with elevated IgG4 levels have inconsistent clinical features are still inconsistent in the current research results.
The immunoturbidimetric assay, electrochemiluminescence immunoassay, and enzyme-linked immunoassay are well known methods for measuring IgG4. Different measurement methods produce inconsistent reference ranges and measured values11,12. Most of the current studies used serum IgG4 ≧ 135 mg/dL as the cut-off value, and inconsistent results may be due to different measurement methods. Masaki et al.13examined the diagnostic cut-off value for IgG4-RD serum IgG4 and discovered that IgG4/IgG ≧ 8% had the highest sensitivity and specificity. As a result, some studies proposed serum IgG4/IgG ≧ 8% as the diagnostic cut-off value for IgG4-RD13,14. However, few studies have been reported on GOs with elevated IgG4 levels, and none have investigated the clinical characteristics of GOs with elevated IgG4 levels before and after treatment using the cut-off value of IgG4/IgG ≧ 8%. Our group previous studies found that IgG4 and IgG4/IgG levels in patients with primary GD were higher than in the normal group and that GD patients with high IgG4/IgG levels had different clinical characteristics than GD patients with low IgG4/IgG levels15.
Therefore, this study first followup newly diagnosed GD patients in the early stages and then used GD patients with no complicated ocular disease as a control group to investigate the relationship between IgG4/IgG and IgG4 levels and the occurrence and development of GO. Next, the clinical and laboratory characteristics of GO patients with elevated IgG4 levels were investigated using the cut-off value of IgG4/IgG ≧ 8%.
Materials and methods
1. Patients and samples
This study enrolled 171 GO patients (111 females and 60 males) who were hospitalized or treated as outpatients in the endocrinology department of the First Affiliated Hospital of Nanchang University from August 2022 to January 2024 as the experimental group. The control group consisted of 57 newly diagnosed GD patients (44 female and 13 male) who had not developed eye disease after a median follow-up of 54.41 months. For patients with active GO, first-line immunosuppressants were primarily used for treatment(4.5 g of glucocorticoids(GC), a full course of 12 weeks (500 mg of GC per week for the first 6 weeks and 250 mg of GC per week for the last 6 weeks). After completing the glucocorticoid treatment course (the third month), outpatient follow-up was conducted to assess the treatment effectiveness.The study enrolled patients who did not receive immunosuppressive therapy and met the diagnostic criteria for GD or GO16, and all patients were ≧ 16 years old and ≦ 65 years old. The patients with the following manifestations were excluded: ①Patients with prior immunosuppressive therapy or optic nerve injury. ② Patients who underwent thyroid surgery or radioactive iodine therapy. ③Individuals with infection, fever, or a history of allergies. ④Individuals with immunodeficiency disorders. ⑤ individuals with other autoimmune diseases or a history of malignancy.⑥Patients with acute or chronic viral hepatitis, moderate to severe liver or kidney impairment, acute infections, severe osteoporosis, high blood pressure, or high glucose.⑦ Individuals who are pregnant or breastfeeding. ⑧ Individuals suspected of or with a history of substance abuse.⑨ Participants who have a history of psychiatric disorders, are deemed unsuitable for the clinical study or are unable to cooperate with its completion, as determined by the investigator. ⑩Participants with a history of mental illness or who are unable to complete the study. This study was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University, and all subjects signed informed consent (Ethics number: 2018-063 and (2022)CDYFYYLK(09002)).
2. Laboratory investigation methods
⑴The serum levels of FT3, FT4, TSH, TPOAb, TGAb, and TRAb were measured using electrochemiluminescence immunoassay.⑵Serum ALT, AST, total bilirubin, and direct bilirubin were detected through automated biochemical analysis.⑶Blood routine was detected by automatic flow cytometry.⑷Serum IgG and IgG4 levels were determined using immunoturbidimetry. The normal reference range is given below: IgG: 7.51–15.60 g/L; IgG4:0.08–1.40 g/L.⑸ Cytokines (IL-6, IL-2, IL-5, IL-10, IL-17, and IFN-Y) were measured using flow cytometry.
3. Methods of assessing ocular conditions
The exophthalmos was measured with a Hertel exophthalmometer. The clinical activity score (CAS) was used to assess ocular activity. A CAS ≧ 3 indicated that the eye disease was active. The Gorman diplopia score was used to evaluate the diplopia of the patients17. The EUGOGO criterion was used to assess eye movement18. Ocular asymmetric lesions are defined as having a binocular protuberance difference of ≧ 2 mm, binocular CAS difference ≧ 2 points, eyelid retraction in one eye, or an ocular motility disorder in one eye.
4. Statistical analysis method
Data analysis was performed using the SPSS 25.0 version and the GraphPad Prism 9.0 software system. Normality between data groups was first determined using Kolmogorov- Smirnov test. Measures that satisfied the normal distribution were expressed as mean ± standard deviation, while those that did not satisfy the normal distribution were expressed as median (P25-P75). For data satisfying normality and variance homogeneity, t-tests were used between the two means, while non-parametric tests were used for data that did not satisfy normal distribution. Count data were compared using the chi-square test. Logistic regression analysis was used to explore the risk factors associated with the occurrence of GO.The correlation between the two variables was investigated using Spearman’s correlation. A paired t-test was used before and after treatment, with P < 0.05 indicating statistical significance.
Results
1. Baseline clinical and laboratory characteristics analysis between GD group and GO group
According to the inclusion and exclusion criteria, 171 GO patients were enrolled in this study as the experimental group (GO group), consisting of 111 women and 60 men. Following a median followup of 54.41 months followup for 84 patients with a primary diagnosis of GD, 57 patients (13 males and 44 females) with GD completed the followup and had no co-morbid ocular disease. In this study, these newly diagnosed GD patients served as the control group (GD group). FT3, FT4, TPOAb, and TGAb were significantly higher in the GD group compared to the GO group. The GO group had a higher number of smokers, TRAb, IgG4, IgG4/IgG, eosinophils, and eosinophils than the GD group, with statistically significant differences. There were no statistically significant differences in gender, age, or IgG between the two groups(Table 1).
Table 1.
Clinical and laboratory characteristics of GD and GO groups.
| GD(n = 57) | GO(n = 171) | P Vaule | |
|---|---|---|---|
| Gender(Male/Femal) | 13/44 | 60/111 | 0.085 |
| Age (years) | 33.00(26.50–43.50) | 37.00(29.00–47.00) | 0.099 |
| Smoking(yes/no) | 7/50 | 43/128 | 0.042* |
| FT3(pg/ml) | 16.28(10.66–25.15) | 3.55(2.91–5.62) | < 0.001*** |
| FT4 (ng/dl) | 5.81(3.63–11.66) | 1.33(0.95–1.95) | < 0.001*** |
| TSH (IU/ml) | 0.003(0.003–0.004) | 0.080(0.005–1.850) | < 0.001*** |
| TPOAb(IU/ml) | 128.30(46.38–305.30) | 42.35(11.15–231.00) | 0.001** |
| TGAb(IU/ml) | 246.30(39.51–576.40) | 17.40(15.00–106.00) | < 0.001*** |
| TRAb(U/L) | 4.90(2.65–14.50) | 8.58(3.47–21.40) | 0.017* |
| IgG (g/L) | 13.20(11.45–15.40) | 11.70(10.30–13.40) | 0.706 |
| IgG4 (g/L) | 0.14(0.05–1.215) | 1.140(0.520–1.930) | < 0.001*** |
| IgG4/IgG(%) | 1.575(0.394–9.176) | 9.652(5.00–14.82) | < 0.001*** |
| Eosinophil count(10^9/L) | 0.09(0.03–0.13) | 0.09(0.06–0.15) | 0.025* |
| Percentage of eosinophils(%) | 1.30(0.50–2.05) | 1.50(1.00–2.70) | 0.024* |
| GD, Graves’ disease. FT3, free triiodothyronine. FT4, free thyroxine. TSH, thyroid-stimulating hormone.TPO-Ab, thyroperoxidase antibody. TG-Ab, thyroglobulin antibody. TRAb, thyrotrophin receptor antibody.IgG, immunoglobulinG.IgG4,immunoglobulinG4. Data are median (25th–75th percentile) unless otherwise indicated. | |||
*p < 0.05; **p < 0.01;***p < 0.001;.
2. Analysis of factors associated with the occurrence of GO
To further investigate the factors related to the development of GO, in this study, the occurrence of eye complications in GD patients was used as the dependent variable, and possible influencing factors such as age, gender, smoking, TPOAb, TGAb, TRAb, IgG4, and IgG4/IgG were used as independent variables in binary logistic regression analysis. The univariate analysis revealed that smoking (P = 0.047), TRAb (P = 0.020), IgG4 (P < 0.001), IgG4/IgG (P < 0.001), and eosinophil level (P = 0.036) were significant influencing factors. We then conducted a multivariate analysis of these factors with significant differences, and after controlling for other significant factors, IgG4/IgG level (OR = 1.362,95% CI = 1.111–1.670, P = 0.003) was found to be an independent influencing factor associated with the occurrence of GO (Table 2).
Table 2.
Univariate and multivariate analyses of GO development.
| Univariate | Multivariate | |||
|---|---|---|---|---|
| OR(95%CI) | P Vaule | OR(95%CI) | P Vaule | |
| Gender | 1.830(0.914–3.662) | 0.088 | - | - |
| Age | 1.023(0.995–1.051) | 0.110 | - | - |
| Smoking | 2.400(1.012–5.688) | 0.047* | 1.471(0.569–3.799) | 0.425 |
| TPOAb | 0.767(0.589–0.998) | 0.081 | - | - |
| TGAb | 0.980(0.967–1.038) | 0.575 | - | - |
| TRAb | 1.559(1.073–2.267) | 0.020* | 1.022(0.996–1.049) | 0.094 |
| IgG | 1.002(0.991–1.012) | 0.723 | - | - |
| IgG4 | 3.273(1.826–5.866) | < 0.001*** | 4.046(0.989–16.550) | 0.052 |
| IgG4/IgG | 4.295(2.300–8.022) | < 0.001*** | 1.362(1.111–1.670) | 0.003** |
| Eosinophil count | 1.472(1.025–2.114) | 0.036* | 7.059(0.132–377.041) | 0.336 |
| Percentage of eosinophils | 1.253(0.991–1.586) | 0.060 | - | - |
| OR, odds ratios.CI, confidence interval | ||||
3. Baseline clinical and laboratory characteristics of GO patients based on IgG4/IgG levels
We divided the enrolled GO patients into two groups: the high IgG4/IgG group (99 cases, 39 males and 60 females) and the low IgG4/IgG group (72 cases in total, including 21 males and 51 females) using the cut-off value of IgG4/IgG ≧ 8%. There were no significant differences in gender or age between the two groups. The number of smokers, TRAb, IgG4, IL-5, IL-17, eosinophils, and eosinophil percentage were greater in the high IgG4/IgG group than in the low IgG4/IgG group, and the difference was statistically significant (P < 0.05), while the probability of binocular symmetrical lesions was substantially higher in the low IgG4/IgG group than in the high IgG4/IgG group. The distinctions in ocular activity status, diplopia, limitation of eye movement, FT3, FT4, TSH, TPOAb, TGAb, IgG, or cytokines (IL-2, IFN-Y, IL-6, and IL-10) were not statistically significant between the two groups (P > 0.05) (Table 3).
Table 3.
Clinical and laboratory characteristics of the high IgG4/IgG and low IgG4/IgG groups in GO patients.
| High IgG4/IgG group(n = 99) | Low IgG4/IgG group(n = 72) | P Vaule | |
|---|---|---|---|
| Gender (Male/Femal) | 39/60 | 21/51 | 0.166 |
| Age (years) | 37.14 ± 11.36 | 38.71 ± 11.57 | 0.378 |
| Ocular lesion activity(yes/no) | 39/60 | 26/46 | 0.662 |
| Diplopia(yes/no) | 24/75 | 21/51 | 0.470 |
| Limited eye movement(yes/no) | 29/70 | 22/50 | 0.859 |
| Binocular symmetry lesion(yes/no) | 61/38 | 56/16 | 0.025* |
| Smoking(yes/no) | 31/68 | 12/60 | 0.029* |
| FT3(pg/ml) | 3.58(2.91–6.62) | 3.48(2.92–5.44) | 0.471 |
| FT4 (ng/dl) | 1.32(1.01–1.95) | 1.34(0.91–1.98) | 0.936 |
| TSH (IU/ml) | 0.070(0.005–1.740) | 0.080(0.005–2.300) | 0.968 |
| TPOAb(IU/ml) | 44.95(10.83–235.30) | 39.70(11.88–147.80) | 0.555 |
| TGAb(IU/ml) | 17.75(15.20–89.80) | 16.80(14.33–165.00) | 0.413 |
| TRAb(U/L) | 10.50(4.19–27.40) | 6.50(2.68–15.75) | 0.047* |
| IgG (g/L) | 12.20(10.50–13.50) | 11.10(10.10–12.95) | 0.099 |
| IgG4 (g/L) | 1.64(1.30–2.31) | 0.48(0.21–0.66) | < 0.001*** |
| IL-2 (pg/ml) | 1.17(0.96–1.45) | 1.11(0.93–1.34) | 0.235 |
| IFN-γ(pg/ml) | 1.34(0.26–3.84) | 1.70(0.36–4.68) | 0.592 |
| IL-5(pg/ml) | 1.45(0.84–2.55) | 1.12(0.67–1.73) | 0.033* |
| IL-6(pg/ml) | 2.33(1.56–4.21) | 1.99(1.23–3.21) | 0.112 |
| IL-10 (pg/ml) | 1.14(0.92–1.65) | 1.14(0.53–1.77) | 0.311 |
| IL-17 (pg/ml) | 1.17(0.23–1.94) | 0.59(0.11–1.64) | 0.031* |
| Eosinophil count(10^9/L) | 0.10(0.07–0.19) | 0.08(0.05–0.13) | 0.013* |
| Percentage of eosinophils(%) | 1.70(1.10–3.10) | 1.30(1.00–2.30) | 0.037* |
|
IL-2,interleukin-2.IFN-γ,interferon-γ.IL-5,interleukin-5.IL-6,interleukin-6.IL-10,interleukin-10. IL-17,interleukin-17. | |||
4. Correlation analysis of serum IgG4, IgG4/IgG, and other clinical indicators in GO patients
To better understand the relationship between serum IgG4 and IgG4/IgG levels and sex, age, ocular lesion characteristics, and laboratory indicators in GO patients, we performed correlation analyses using Spearman correlation analysis. The results showed that TRAb, eosinophils, eosinophil percentage, and IL-5 were found to have a significant and positive correlation with IgG4/IgG and IgG4 levels (P < 0.05). In contrast, ocular symmetric lesions in GO patients showed a significant negative correlation with IgG4/IgG (r = − 0.156, P = 0.042) and IgG4 (r = − 0.155, P = 0.043) levels. We also discovered a significant positive correlation between smoking and IgG4/IgG levels (r = 0.168, P = 0.028), but no significant correlation was observed with IgG4 levels (P > 0.05). Gender, age, FT3, FT4, TSH, TPOAb, TGAb, and cytokines (IL-2, IFN-Y, IL-6, IL-10, and IL-17) had no significant relationship with IgG4/IgG and IgG4 levels (Table 4).
Table 4.
Correlation analysis of serum IgG4, IgG4/IgG and other clinical indicators in GO patients.
| IgG4/IgG | IgG4 | |||
|---|---|---|---|---|
| R | P | R | P | |
| Gender | 0.145 | 0.059 | 0.066 | 0.389 |
| Age | −0.113 | 0.142 | −0.085 | 0.268 |
| Smoking | 0.168 | 0.028* | 0.084 | 0.273 |
| Binocular symmetry lesion | −0.156 | 0.042* | −0.155 | 0.043* |
| FT3(pg/ml) | 0.044 | 0.566 | 0.025 | 0.741 |
| FT4 (ng/dl) | 0.003 | 0.974 | 0.007 | 0.932 |
| TSH (IU/ml) | −0.028 | 0.714 | −0.046 | 0.552 |
| TPOAb(IU/ml) | 0.064 | 0.424 | 0.067 | 0.401 |
| TGAb(IU/ml) | 0.025 | 0.753 | 0.049 | 0.534 |
| TRAb(U/L) | 0.155 | 0.043* | 0.158 | 0.039* |
| IL-2 (pg/ml) | 0.071 | 0.359 | 0.075 | 0.330 |
| IFN-γ(pg/ml) | −0.08 | 0.3 | −0.038 | 0.625 |
| IL-5(pg/ml) | 0.172 | 0.025* | 0.170 | 0.027* |
| IL-6(pg/ml) | 0.061 | 0.433 | 0.101 | 0.190 |
| IL-10 (pg/ml) | 0.093 | 0.227 | 0.096 | 0.215 |
| IL-17 (pg/ml) | 0.090 | 0.244 | 0.125 | 0.106 |
| Eosinophil count(10^9/L) | 0.258 | 0.001** | 0.246 | 0.001** |
| Percentage of eosinophils | 0.241 | 0.002** | 0.227 | 0.003** |
5. Clinical and laboratory characteristics analysis of GO patients after treatment
Thirty-two patients (16 males and 16 females) with moderately severe active GO completed the treatment course and 3-month followup. After treatment, patients with GO had lower CAS, TPOAb, TRAb, IgG, and percentage of eosinophils compared to those before treatment, with a statistically significant difference (p < 0.05). There were no statistically significant differences in FT3, FT4, TSH, TGAb, IgG4, IgG4/IgG, eosinophils, or cytokines (IL-2, IFN-Y, IL-5, IL-6, IL-10 and IL-17) between the two groups (P > 0.05) (Table 5).
Table 5.
Clinical and laboratory characteristics of GO patients before and after treatment (n = 32).
| Before treatment | After treatment | P Vaule | |
|---|---|---|---|
| CAS | 3.50(2.62–4.00) | 2.00(2.00–2.88) | < 0.001*** |
| FT3(pg/ml) | 3.57(2.89–4.98) | 3.19(2.90–3.70) | 0.178 |
| FT4 (ng/dl) | 1.33(0.84–1.81) | 1.32(1.16–1.53) | 0.807 |
| TSH (IU/ml) | 0.075(0.006–3.210) | 1.630(0.390–2.883) | 0.262 |
| TPOAb(IU/ml) | 18.40(5.65–70.68) | 15.70(4.50–33.40) | 0.004** |
| TGAb(IU/ml) | 16.85(14.85–29.50) | 16.80(14.10–22.30) | 0.016* |
| TRAb(U/L) | 7.50(3.53–21.78) | 2.66(1.36–8.76) | < 0.001*** |
| IgG (g/L) | 10.75(9.36–12.30) | 9.48(8.92–10.80) | < 0.001*** |
| IgG4 (g/L) | 1.10(0.56–2.18) | 0.92(0.46–1.69) | 0.583 |
| IgG4/IgG(%) | 9.83(5.54–16.33) | 9.76(4.72–20.55) | 0.295 |
| IL-2 (pg/ml) | 1.27 ± 0.42 | 1.17 ± 0.33 | 0.228 |
| IFN-γ(pg/ml) | 0.82(0.23–1.46) | 0.98(0.05–1.91) | 0.336 |
| IL-5(pg/ml) | 1.21(0.92–2.73) | 1.41(0.88–3.45) | 0.266 |
| IL-6(pg/ml) | 1.97(1.35–3.25) | 1.69(0.81–2.54) | 0.130 |
| IL-10 (pg/ml) | 1.16(0.88–1.57) | 1.15(0.85–1.64) | 0.851 |
| IL-17 (pg/ml) | 0.81(0.16–1.64) | 0.78(0.37–1.54) | 0.612 |
| Eosinophil count(10^9/L) | 0.10(0.07–0.16) | 0.09(0.06–0.13) | 0.275 |
| Percentage of eosinophils(%) | 2.00(1.20–3.20) | 1.30(0.80–2.40) | < 0.001*** |
Discussion
Our findings revealed significantly higher levels of IgG4 and IgG4/IgG in GO patients compared to GD patients, but after controlling for other factors, we discovered that the IgG4/IgG level was the independent influencing factor associated with the occurrence of GO. Although IgG4/IgG is linked to the development of GO, there was no significant difference in ocular activity status between GO patients with high IgG4/IgG levels and the group with low IgG4/IgG levels in this study. Furthermore, while the activity score of eye disease decreased after treatment, neither IgG4 nor IgG4/IgG showed a significant reduction. Therefore, we believe that IgG4/IgG levels are not significantly associated with ocular activity in GO patients. In this study, 57.89% (99/171) of the GO patients had high serum IgG4/IgG levels. These patients with high IgG4/IgG GO exhibit different characteristics than those with low IgG4/IgG GO. They had significantly higher rates of smokers, TRAb, IgG4, IL-5, IL-17, eosinophils, and eosinophil percentages, and were more likely to have asymmetric lesions in both eyes.
The thyroid is one of the organs involved in IgG4-RD, accounting for approximately 5.6%2. Currently, research data on GD and GO with elevated IgG4 levels is limited, and the findings are contradictory19,20. In 2017, Yu7et al. examined the clinical characteristics of 64 patients with GD and discovered that IgG4 levels (reference range: 3.9–86.4 mg/dL) were independently correlated with the occurrence and grading of GO. This supports the findings of Bozkirli et al.21in 2015. Olejarz8, Ye9, and Zhao22et al. also discovered that serum IgG4 levels were linked to ocular activity in GO patients. In contrast, a retrospective analysis of 297 GD patients conducted by Comi10et al. in 2023 discovered that IgG4 levels were not associated with the occurrence and progression of GO. The above studies used different measurement methods and reference ranges for IgG4, as well as different study types, which could explain the inconsistent results. Consistent with the findings of Yu7and Bozkirli21 et al., it was discovered in this study that the levels of IgG4 and IgG4/IgG in GO patients were significantly higher than those in GD patients, but IgG4/IgG was only independently correlated with GO only after adjusting for other influencing factors. This study found no correlation between IgG4 and IgG4/IgG levels and GO activity status.
T lymphocytes and their secreted cytokines are essential for regulating the body’s immune balance, according to studies23. The findings of this study revealed that the high IgG4/IgG level GO group had significantly higher IL-5 levels than the low IgG4/IgG level GO group. It is well understood that IL-5 is a major cytokine secreted by Th2 lymphocytes24. Studies have shown that cytokines secreted by Th2 cells can promote the production of IgG4 by B lymphocytes as well as the production of eosinophils25. Olejarz8et al. and Torimoto26 et al. found that eosinophils in GO or GD patients with elevated IgG4 levels were significantly increased, which was consistent with the findings of this study. This study also found that the percentage of eosinophils in the GO group with high IgG4/IgG levels was also significantly increased. They also showed a positive correlation with IgG4/IgG and IgG4 levels. IL-17 is the main cytokine secreted by Th17 cells. Studies have shown that an increase in the production of Th17 cell subpopulation or its secretion of cytokines is linked to the development of autoimmune diseases. In this study, IL-17 levels were significantly higher in the GO group with high IgG4/IgG levels, indicating that there was a lymphocyte subset imbalance in the GO group with high IgG4/IgG levels.
TRAb is a significant influencing factor in the occurrence and progression of GO patients27. TRAb was previously thought to be made up entirely of IgG128. However, Latrofa et al.29 found that long-term antigenic stimulation can increase TRAb concentration and eventually convert IgG1 to IgG4 in GD patients. The study results of Takeshima et al.30 in 2014 discovered that the titer of TRAb in GD patients with elevated serum IgG4 levels was positively correlated with IgG4. Furthermore, Yu et al.7reported in 2017 that the level of IgG4 was correlated with the level of TRAb in GD patients with GO. Consistent with the findings of Yu et al.7, this study discovered a positive correlation between IgG4 level and TRAb level in GO patients, as well as a positive correlation between IgG4/IgG and TRAb level. In multifactor analysis, IgG4/IgG was identified as an independent influence factor on GO. Thus, our findings support measuring IgG4 and IgG4/IgG levels to predict the occurrence of GO.
It is well known that smoking is harmful to one’s health, and in patients with GD, smoking is one of the risk factors contributing to the development of GO, but the exact mechanism has yet to be determined31. A large-scale health screening cohort study conducted by Tsuj32et al. in 2023 discovered a positive correlation between smoking and IgG4 levels. Tsai33and Liao34et al. demonstrated that smoking induces IgG4-RD. In this study, it was discovered that the smoking rate in GO patients was significantly increased, and there was a significant positive correlation between smoking and IgG4/IgG in GO patients, implying that the mechanism by which smoking promotes GO may be related to the promotion of IgG4 levels and the increase of IgG4/IgG levels. GO is typically bilateral, but approximately 9–15% of patients with GO have only unilateral involvement35. Bilateral asymmetric lesions are even more common, accounting for up to 36%36–38. Luo39 et al. examined eight cases of GO patients in combination with eyelid swelling degree, ocular projection degree, and ocular muscle characteristics, and found that the ocular lesions of GO patients with elevated IgG4 levels had bilateral symmetry. However, in this study, 171 patients with GO were studied, and the ocular lesions of GO patients with elevated IgG4/IgG levels were discussed in conjunction with the characteristics of ocular exophthalmia, eyelid retraction, CAS score, and eye movement in GO patients. The findings revealed that the incidence of binocular asymmetry in GO patients with high IgG4/IgG levels was significantly higher than in the low IgG4/IgG level group. IgG4 and IgG4/IgG levels showed a positive correlation with bilateral asymmetrical lesions. Currently, no studies on IgG4/IgG levels and ocular lesion characteristics in patients with GO have been reported.
There are several limitations to this study: First, the patient did not undergo surgery or thick needle puncture; as such, no pathological results of the thyroid were obtained. Second, the sample size in this study is not particularly large, which necessitates further investigation and verification through larger sample sizes.Third, In this study, only 3 months of followup was conducted, which is insufficient for a more comprehensive dynamic observation of the changes in IgG4 and IgG4/IgG levels with changes in the condition after treatment in patients with GO, and future studies should extend the followup period.
Conclusions
In conclusion, our findings indicate that serum IgG4/IgG levels are independently associated with the occurrence of GO, but not with the state of ocular activity in GO. Patients with high IgG4/IgG levels of GO exhibit distinct characteristics. They are distinguished by a high smoking rate, TRAb, IL-5, IL-17, and percentage of eosinophils, as well as a proclivity for binocular asymmetrical lesions, which may be associated with an unstable balance of lymphocyte subsets.
Acknowledgements
We are grateful to the Clinical Laboratory of the First Affiliated Hospital of Nanchang University for the support of the detection technology of this study.
Abbreviations
- GO
Graves’ ophthalmopathy
- GD
Graves’ disease
- FT3
Free triiodothyronine
- FT4
Free thyroxine
- TSH
Thyroid-stimulating hormone
- TPO-Ab
Thyroperoxidase antibody
- TG-Ab
Thyroglobulin antibody
- TRAb
Thyrotrophin receptor antibody
- IL-6
Interleukin-6
- IL-2
Interleukin-2
- IL-5
Interleukin-5
- IL-10
Interleukin-10
- IL-17
Interleukin-17
- IFN-γ
Interferon-γ
- CAS
Clinical activity score
- GC
Glucocorticoids
Author contributions
Y.D. and J.X.conceptualized and design the study. Y.D.,Z.H.,S. L. ,J.Z.and J.Z.contributed to collection of data and specimens. Y.D.and J.X.contributed to analyzed the data and drafted the manuscript. Y.D.,H.G.,W.C.,Y.L. and J. X.reviewed and edited the manuscript. All authors reviewed the manuscript.
Funding
The manuscript was supported by grants from The Natural Science Foundation of Jiangxi Province [grant numbers. 20224ACB206010], Construction of scientific and technological Innovation base - Clinical medical Research Center [grant no. 20221ZDG02011], The National Natural Science Foundation of China [grant no. 82160140], and The Young Talent Research and Development Program of the First Affiliated Hospital of Nanchang University[grant no. YFYPY202410].
Data availability
The data will be shared on reasonable request to the corresponding authors.
Declarations
Competing interests
The authors declare no competing interests.
Ethics approval
This study involving human participants was in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards, and was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University (Ethics number: 2018-063 and (2022)CDYFYYLK(09002)).
Informed consent
Informed consent was obtained from patient’s or legal guardians.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
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Data Availability Statement
The data will be shared on reasonable request to the corresponding authors.