The titers of antinuclear antibodies are associated with the degree of inflammation and organ damage in Primary Sjögren's Syndrome

The study included patients with pSS treated at the First Affiliated Hospital of the University of Science and Technology of China from July 2019 to May 2023. The diagnosis of enrolled patients adhered to the classification criteria for pSS jointly established by the ACR and EULAR in 2016 [5, 6]. This study has received approval from the Ethics Committee of the First Affiliated Hospital of the University of Science and Technology of China, with the ethics approval number: 2023-RE-279. Furthermore, all patients underwent lip biopsy at our hospital, and lip gland pathology was categorized as grade 2 or above, and all have signed informed consent forms. Exclusion criteria for enrolled patients were as follows: (1) Presence of other connective tissue diseases besides Sjögren’s Syndrome, such as systemic lupus erythematosus or dermatomyositis; (2) Patients during pregnancy or lactation; (3) Patients with severe infections.

Retrospective collection and organization of patients' general clinical information, complications, and laboratory examination data. The main laboratory indicators include ANA titers, Extractable Nuclear Antigen Antibodies (ENA) profile (including anti-SSA 52 antibodies, anti-SSA 60 antibodies, anti-SSB antibodies), tear flow rate (left and right), salivary flow rate, immunoglobulin (Ig) levels(A、G and M), complement (C) levels, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), white blood cell count (WBC), hemoglobin (Hb), platelet count (PLT), rheumatoid factor (RF), lip gland pathology, and organ involvement.

The primary methods for laboratory indicator testing are as follows: ANA is detected using an indirect immunofluorescence method with EUROIMMUN reagents from Germany. ENA is detected using EUROIMMUN immunoblotting. ESR is measured using the ALIFAX Test 1, a fully automated rapid sedimentation analyzer from ALIFAX, Italy. CRP, RF, Ig, C3, C4, and liver function are assessed using immunoturbidimetric assays with reagents from SIEMENS, Germany. WBC, Hb, and PLT are measured using a resistance-type blood analyzer from Mindray, China. Chest examination is conducted using High-Resolution Computed Tomography with a Siemens Force CT scanner from Siemens, Germany. Microscopic analysis is performed on surgically excised minor salivary gland lobules under local anesthesia to obtain salivary gland pathology results, which are graded according to Chisholm's criteria into grades I-IV [11].

Tear flow rate testing method: A 5 mm by 35 mm strip of filter paper is inserted into the inner third of the lower eyelid conjunctival sac, with 5 mm of one end folded and the rest hanging over the skin surface. After gently closing the eyes for 5 min, the length of paper moistened by tears is measured, with less than 10 mm considered abnormal.

Salivary flow rate testing method: Take a clean, dry cotton ball and record its weight. The individual expels their saliva, then inserts the cotton ball into the posterior molar region, chews for 2 min, and subsequently spits it out along with saliva. Measure the weight of the cotton ball (including saliva) and compare the difference in weight before and after; less than 2 g is considered abnormal.

Initially, patients were categorized into two groups based on antinuclear antibody (ANA) titers: the ANA-positive group (ANA ≥ 1:320) and the ANA-negative group (ANA < 1:320). A comparison was conducted between these groups, considering general demographic data, laboratory indicators, lip gland pathology grading, and organ involvement. Subsequently, to explore the potential association between ANA titer stratification and multi-organ damage, a comparison was made among patients with different ANA titers, considering the same parameters. During the study, it was observed that some ANA-negative patients could still test positive for anti-SSA and anti-SSB antibodies. Consequently, patients were further divided based on ANA titers and the positivity or negativity of anti-SSA and anti-SSB antibodies into the positive group (at least one positive result among ANA titers, anti-SSA, and anti-SSB antibodies) and the negative group (negative results for all ANA titers, anti-SSA, and anti-SSB antibodies), and a comparison was made regarding their baseline characteristics. Lastly, recognizing the sensitivity and specificity of anti-SSA and anti-SSB antibodies for Sjögren's Syndrome, within the positive group, patients were further classified into the anti-SSA positive group, anti-SSB positive group, both positive group, and both negative group based on the positivity or negativity of anti-SSA and anti-SSB antibodies. A comparison was then conducted among these four groups, considering general demographic data, laboratory indicators, lip gland pathology grading, and organ involvement.

Criteria for organ involvement: According to the "Diagnosis and Treatment Guidelines for Primary Sjögren's Syndrome" [12], approximately one-third of Sjögren's Syndrome patients may experience systemic involvement, mainly affecting the skin, joints and muscles, respiratory system, digestive system, kidneys, nervous system, blood system, cryoglobulinemia, and autoimmune thyroid diseases. However, in the patients included in this study, there were no neurological involvement or cryoglobulinemia. Therefore, only the seven types of systemic involvement mentioned above were studied.

Statistical analysis of the data was conducted using SPSS 25.0 software. For normally distributed and homogenous data, mean ± standard deviation (\(\overline{x} \pm s\)) was employed to represent quantitative variables, and t tests were utilized for comparisons between two groups. Non-normally distributed quantitative variables were presented as median [M (P25, P75)], and nonparametric rank-sum tests were applied for comparisons between two or more groups. Qualitative variables were expressed as percentages (%), and chi-square tests were employed for comparisons between groups. A significance level of P < 0.05 was considered statistically significant. For missing data less than 30%, missing values were replaced to complete the data. In cases of missing data exceeding 30%, multiple imputation was used to supplement the missing data.

A total of 551 patients were included, with a male-to-female ratio of 1: 35.7, and the age of onset ranged from 14 to 85 years, with a peak incidence between 40 and 60 years. Among them, there were 443 cases in the positive group for ANA, with 18 males and 425 females and an average age of 49.35 ± 13.11 years. In the negative group for ANA, there were 108 cases, with 1 male and 107 females, and an average age of 51.62 ± 12.02 years. According to the titers of ANA, the patients were divided into 5 groups: Group 1 (ANA titer < 1:320) with 106 cases, 6 males and 100 females, and an average age of 51.44 ± 12.02 years; Group 2 (1:320 ≤ ANA titer < 1:1000) with 94 cases, 5 males and 89 females, and an average age of 49.35 ± 11.62 years; Group 3 (1:1000 ≤ ANA titer < 1:3200) with 170 cases, 5 males and 165 females, and an average age of 48.16 ± 13.62 years; Group 4 (1:3200 ≤ ANA titer < 1:10,000) with 135 cases, 2 males and 133 females, and an average age of 50.99 ± 13.33 years; Group 5 (ANA titer ≥ 1:10,000) with 45 cases, 5 males and 40 females, and an average age of 50.27 ± 12.08 years. Based on the antibody status, the patients were divided into a positive group (those with positive ANA, anti-SSA, or anti-SSB antibodies) and a negative group (those with negative ANA, anti-SSA, and anti-SSB antibodies). The positive group consisted of 531 cases, with 22 males and 509 females, and an average age of 49.74 ± 12.88 years. The negative group had 19 cases, with 1 male and 18 females, and an average age of 53.26 ± 10.95 years. Among the positive group, further subgroups were defined based on the presence or absence of anti-SSA and anti-SSB antibodies: anti-SSA positive (301 cases, 14 males and 287 females, average age: 49.60 ± 12.62 years), anti-SSB positive (2 cases, 0 males and 2 females, average age: 42.00 ± 11.31 years), both positive (167 cases, 6 males and 161 females, average age: 48.54 ± 14.03 years), and both negative (80 cases, 3 males and 77 females, average age: 53.81 ± 10.04 years).

Among the initial symptoms, dry mouth was the most common, occurring in approximately 361 patients (65.52%). The second most common symptom was a combination of dry mouth and dry eyes, seen in approximately 235 cases (42.65%). During the course of the disease, dental caries were observed in about 109 patients (19.78%) and arthralgia in 149 cases (27.04%). Among the complications, leukopenia was the most common, occurring in approximately 163 cases (29.58%), followed by pulmonary interstitial lesions in about 120 cases (21.78%). Other complications included liver dysfunction in about 109 cases (19.78%), thyroid abnormalities in about 95 cases (17.24%), primary biliary cirrhosis in 21 cases (3.81%), and renal tubular acidosis in about 12 cases (2.18%).

Based on the analysis of antinuclear antibody spectrum in the included patients, the highest positivity rate was found for anti-SSA52 antibody, with approximately 422 cases (76.59%), followed by anti-SSB antibody with 390 cases (70.78%). There were 80 cases (14.52%) negative for both anti-SSA and anti-SSB antibodies. Among the positive cases, 163 cases (29.58%) were positive for both anti-SSA52, anti-SSA60, and anti-SSB antibodies. Among other related antibodies, positive rates were seen for anti-CENP B antibodies in 88 cases (15.97%) and anti-cellular antibodies in 79 cases (14.34%).

All included patients underwent lip gland biopsy and pathological examination. After local disinfection and local anesthesia, 3–5 lip gland samples were taken from the lower lip mucosa, and the incision was sutured. The gland specimens were fixed in a 10% formaldehyde solution, embedded, sectioned, and stained with hematoxylin and eosin (HE). The pathology slides were observed under a microscope. According to the Chisholm criteria and the degree of lymphocyte infiltration (LC), lip gland pathology changes were divided into 0–4 grades: Grade 0 indicated no LC infiltration, Grade 1 indicated mild LC infiltration, Grade 2 indicated moderate LC infiltration without formation of focal lesions, Grade 3 indicated the presence of one LC infiltration focus in an area of ≥ 4 mm², and Grade 4 indicated the presence of multiple LC infiltration foci in an area of ≥ 4 mm², with each focus containing 50 lymphocytes and tissue cells. The number of lymphocytic foci in lip gland specimens was counted. According to the Chisholm grading criteria, among the included patients, 145 cases (26.32%) were Grade 2, 155 cases (28.13%) were Grade 3, and 251 cases (45.55%) were Grade 4.

Through the comparison (as seen in Fig. 1), we found that among patients with negative antinuclear antibody titers and positive antinuclear antibody titers, the laboratory indicators of patients in the positive antinuclear antibody titer group were higher for ESR, RF, IgA, and IgG compared to the negative antinuclear antibody titer group. The differences between the two groups were statistically significant (all P < 0.05). Additionally, the tear flow rate (left and right), saliva flow rate, complement C3, complement C4, and WBC count were lower in the positive antinuclear antibody titer group compared to the negative antinuclear antibody titer group, and these differences were statistically significant (all P < 0.05). Furthermore, upon further analysis, we found that the pathological grades of labial salivary gland biopsies and the number of organ involvement were higher in the positive antinuclear antibody titer group compared to the negative antinuclear antibody titer group, and these differences were statistically significant (all P < 0.05). In terms of organ involvement (as seen in Table 1), we found that the rates of skin and mucosal and hematological system involvement were generally higher in the positive antinuclear antibody titer group compared to the negative antinuclear antibody titer group, and these differences were statistically significant (all P < 0.05). However, there was no statistical significance between the two groups in terms of rates of musculoskeletal, urinary, respiratory, digestive system, and thyroid involvement (all P > 0.05). In various organs, there are some commonly observed immune-related injuries, such as immune-related interstitial pneumonia in the respiratory system, renal tubular acidosis in the urinary system, autoimmune hepatitis in the digestive system, and immune thrombocytopenia in the blood system (physiological decreases in white blood cell and hemoglobin levels are present, and multiple influencing factors are involved, immune-related factors may not be the dominant cause, therefore not explored in this study). We analyzed the immune-related injuries in this specific patient group and found that there were no statistically significant differences between the two groups (all P > 0.05).

Based on the patient's antinuclear antibody titer, we divided them into five groups: Group 1 (antinuclear antibody titer < 1:320), Group 2 (1:320 ≤ antinuclear antibody titer < 1:1000), Group 3 (1:1000 ≤ antinuclear antibody tite < 1:3200), Group 4 (1:3200 ≤ antinuclear antibody titer < 1:10,000), and Group 5 (antinuclear antibody titer ≥ 1:10,000). We compared the general demographic and laboratory characteristics, labial salivary gland pathology grades, and organ involvement between these groups. The results (as seen in Fig. 2) indicated that as the antinuclear antibody titer increased, there were differences in the levels of immunoglobulin IgG and the number of affected organs among different antinuclear antibody titer levels, and these differences were statistically significant (P < 0.05). We also found that with increasing antinuclear antibody titer levels, there was an increasing trend in ESR, RF, and salivary flow rate (left), but only the difference between Group 5 (antinuclear antibody titer ≥ 1:10,000) and Group 1 (antinuclear antibody titer < 1:320) was statistically significant (P < 0.05). There were no statistically significant differences between the other groups. Furthermore, as the antinuclear antibody titer level increased, the levels of complement C3 and complement C4 showed a decreasing trend. Only the difference between Group 3 (1:1000 ≤ antinuclear antibody titer < 1:3200) and Group 1 (antinuclear antibody titer < 1:320) was statistically significant (P < 0.05), while there were no statistically significant differences between the other groups. Regarding salivary flow rate, with increasing antinuclear antibody titer levels, there was a general trend of decreased salivary flow rate in patients. The differences between Group 3 (1:1000 ≤ antinuclear antibody titer < 1:3200), Group 4 (1:3200 ≤ antinuclear antibody titer < 1:10,000), and Group 1 (antinuclear antibody titer < 1:320) were statistically significant (P < 0.05), while there were no statistically significant differences between the other groups. There were no significant differences in CRP, IgA, IgM, WBC, Hb, PLT, and pathological grades among the groups, and these differences were not statistically significant. In terms of organ involvement (as seen in Table 2), with an increase in antinuclear antibody titer levels, there was an overall upward trend in the positive rates for skin/mucous membrane and the hematological system, and these differences were statistically significant (P < 0.05). However, there were no significant statistical differences observed in other organ involvement rates. And we analyzed the immune-related injuries of specific organs between the two groups and found no statistical difference between the two groups (all P > 0.05).

In our study, we further discovered that some patients with negative antinuclear antibody titers tested positive for anti-SSA and anti-SSB antibodies. We reclassified the 551 collected patients into two groups: the positive group (patients with at least one positive result for antinuclear antibody titer, anti-SSA, or anti-SSB antibodies) and the negative group (patients with negative results for antinuclear antibody titer, anti-SSA, and anti-SSB antibodies). We compared the general demographic and laboratory characteristics, labial salivary gland pathology grades, and organ involvement between these two groups. The results (as seen in Tables 3 and Fig. 3) showed that in terms of laboratory tests, the negative group had significantly lower IgG levels compared to the positive group (P < 0.05). The negative group also had significantly higher complement C4 and platelet levels compared to the positive group (P < 0.05). Although the negative group generally had lower levels of RF, CRP, and IgA compared to the positive group, and higher tear and salivary flow rates, white blood cell counts, and hemoglobin levels, these differences were not statistically significant (P > 0.05). Additionally, the number of affected organs in the negative group was generally lower than that in the positive group, but this difference was not statistically significant (P > 0.05). The rate of organ involvement and the rate of autoimmune injury of specific organs were further compared between the two groups, and there was no statistical significance (P > 0.05).

We then hypothesized that anti-SSA and anti-SSB antibodies, as the most sensitive and specific antibodies for pSS, may have an impact on the laboratory indicators and organ damage in patients. To further investigate this, we compared the positive group mentioned above based on the positivity of anti-SSA and anti-SSB antibodies. The groups were categorized as the anti-SSA positive group, anti-SSB positive group, double-positive group, and double-negative group. The results are as follows (as seen in Tables 4 and Fig. 4): We found differences among the groups in terms of ESR, RF, IgA, IgG, IgM, complement C3, WBC, Hb, and pathological grades. Upon pairwise comparisons, significant differences were observed in ESR levels between the double-positive group and the anti-SSA positive group as well as the double-negative group (adjusted P = 0.001, P = 0.009). Significant differences were also found in RF levels between the double-positive group and the anti-SSA positive group, as well as the double-negative group (adjusted P < 0.001, P = 0.009). In terms of IgA levels, there were significant differences between the double-positive group and the double-negative group (adjusted P = 0.007). Significant differences were observed in IgM levels between the double-positive group and the anti-SSA positive group, as well as the double-negative group (adjusted P = 0.003, P = 0.001). For IgG levels, significant differences were found between the double-positive group and the anti-SSA positive group, anti-SSB positive group, and the double-negative group (adjusted P < 0.001, P = 0.008, P < 0.001). Complement C3 levels showed significant differences between the double-positive group and the double-negative group (adjusted P = 0.049). Hemoglobin levels had significant differences between the double-positive group and the anti-SSA positive group, as well as the double-negative group (adjusted P = 0.002, P = 0.002). White blood cell counts showed significant differences between the double-positive group and the anti-SSA positive group, as well as the double-negative group (adjusted P = 0.001, P < 0.001). In terms of pathological grades, significant differences were observed between the double-positive group and the anti-SSA positive group (adjusted P = 0.027). In terms of organ involvement, we found that the positive anti-SSA antibody may be related to the blood system injury and the occurrence of autoimmune hepatitis, and the difference was statistically significant (P < 0.05).