Primary Sjögren’s syndrome (pSS) is a slowly progressed autoimmune disorder characterized by lymphocytic infiltration of exocrine glands and subsequent significant loss of secretory function with oral or eye dryness [
Natural killer (NK) cells are innate lymphoid cells that exhibit the capacity to secrete cytokines and possess natural cytotoxicity [
NK cells are characterized conventionally by the expression of the CD56 surface marker [
In this study, we analyzed the characteristics of peripheral blood CD56bright NK cell subset and CD56dim NK cell subset in patients with pSS and found that the ratio of CD56bright NK to CD56dim NK was elevated, and the area under the curve (AUC) of this ratio was 0.838 by receiver operator characteristic (ROC) curve analysis. Furthermore, this ratio was positively associated with serum IgG level and negatively associated with complement C3 and C4 levels, though not associated with EULAR Sjögren’s Syndrome Disease Activity Index (ESSDAI) in pSS patients.
Independent identification and newly diagnosed patients with pSS, SLE, and IgG4-related disease (IgG4-RD) as well as healthy controls (HC) were included in this study. Samples of patients were obtained from the clinic of the Department of Rheumatology and Immunology, Tongji Hospital from November 2017 to November 2018. Patients with pSS (52), SLE [
The approval of the ethical committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology was obtained before the study. The ethical institutional review board (IRB) ID is TJ-C20151109. All subjects have provided written informed consent to participate in the study.
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood samples from all patients and controls by Ficoll-Paque (GE Healthcare, Amersham, UK) density gradient centrifugation. Fluorochrome-conjugated antibodies specific for the following cell surface molecules were used: anti-human-CD3 (HIT3a)-PE and anti-human-CD56 (HCD56)-FITC (antibody reagents were purchased from Biolegend, UK). PBMCs were incubated with fluorochrome-conjugated antibodies diluted in PBS for cell surface staining at 4°C. Cells were subsequently washed and resuspended in PBS prior to analysis. Flow cytometry was carried out by using a Calibur flow cytometer (BD Biosciences, San Diego, CA), and data were analyzed using FlowJo 7.6 software (Tree Star, Inc., Ashland, OR). The lymphocyte population was identified by assessment of the size and granularity of cells using forward scatter (FSC) and side scatter (SSC). NK cell percentage expressed as a proportion of total gated lymphocytes. Among NK cells, CD56bright and CD56dim subsets were identified according to the level of CD56 expression. Gates were set by using isotype control antibodies.
GraphPad Prism 5.0 software was used for all statistical analyses. The Mann-Whitney
The demographic characteristics and clinical features of pSS patients and healthy controls are described in Table
Overview of individuals at baseline used in this study.
pSS | HC | |
---|---|---|
Characteristics | ||
Number | 52 | 20 |
Age in years ( | ||
Range | 20-68 | 24-77 |
Female : male | 49 : 3 | 19 : 1 |
Clinical features | ||
ANA+ (%) | 44 (84.6) | 0 (0) |
Ro/SSA+ (%) | 44 (84.6) | NA |
La/SSB+ (%) | 23 (44.2) | NA |
ESR, high levels (>20 mm/h) (%) | 25 (48.1) | NA |
CRP, high levels (>5 mg/l) (%) | 7 (13.5) | NA |
IgG, elevated levels (>16 g/l) (%) | 26 (50) | NA |
Focus | 42 (87.5)a | NA |
ESSDAI range (median) | 0-15 (4) | NA |
Categorical data are expressed as the absolute frequency, with percentages in parenthesis. ANA: antinuclear antibodies; Ro/SSA+: antibodies against Ro/Sjögren’s syndrome A antigen; La/SSB+: antibodies against La/Sjögren’s syndrome B antigen; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein; focus score indicates the number of inflammatory foci containing more than 50 mononuclear cells per 4 mm2 biopsy tissue. ESSDAI: EULAR Sjögren’s Syndrome Disease Activity Index; NA: not applicable. a
In accordance with previous literature [
Comparison of percentage and counts of peripheral blood NK cells and CD56 NK cell subsets between pSS patients and HC. (a) The gating strategy is shown for the analysis of NK population. (b) The percentage of peripheral blood NK cells and their constituent CD56 subsets. (c) The absolute number of peripheral blood NK cells and their constituent CD56 NK cell subsets. (d) The ratio of CD56bright NK/CD56dim NK in autoimmune diseases. The horizontal line represents the mean. Definitions of abbreviations: HC = healthy controls; pSS = primary Sjögren’s syndrome.
We further calculated the ratio of CD56bright NK cell subset to CD56dim NK cell subset as described above and found that the ratio of CD56brightNK to CD56dimNK was significantly elevated in pSS patients than that in healthy controls (
ROC curve analysis indicated that the area under the curve (AUC) was 0.838, and the best diagnostic cut-off point of the ratio of CD56bright NK to CD56dim NK was 0.0487 for the patients with pSS (Figure
ROC curve of the ratio of CD56bright NK to CD56dim NK. The curve plots the relationship between sensitivity and 1 − specificity for different cut-off levels. When 0.0487 was used as the cut-off point for the diagnostic score of suspected pSS patients, the maximum value of the Youden index was achieved as 0.596.
We next analyzed the correlation of CD56bright NK to CD56dim NK ratio with disease activity in pSS patients. Spearman rank correlation did not identify a significant correlation of the ratio with the ESSDAI (supplementary figure
The correlation between CD56bright NK/CD56dim NK ratio and disease activity. (a) The correlation analysis between the CD56bright NK/CD56dim NK ratio and the serum IgG level or (b) complement C3 level or (c) complement C4 value in pSS patients.
We have compared the CD56bright NK, CD56dim NK, and CD56bright NK to CD56dim NK ratio between before and after treatment in pSS patients. The drugs of treatment are mainly glucocorticoid and hydroxychloroquine. As shown in supplementary figure
Increasing experimental evidence has shown a direct involvement of NK cells and CD56 NK cell subsets in some human immunopathologies such as psoriatic arthritis, SLE, multiple sclerosis, and Behcet’s disease [
As expected, we observed that the CD56bright NK to CD56dim NK ratio had the potential to diagnose pSS in autoimmune diseases. Using a cut-off value of 0.0487, we found that the sensitivity and specificity of the ratio were 84.6% and 75%, respectively. In order to further determine the significance of the CD56bright NK to CD56dim NK ratio in the identification of pSS patients, the changes of this ratio in other autoimmune diseases were analyzed. Accordingly, we found that the CD56bright NK to CD56dim NK ratio was also increased in SLE patients and unchanged in IgG4-RD. These distinct changes of the ratio in different autoimmune diseases may be attributed to their different pathological mechanisms. It has been reported that much similar pathogenesis have existed between patients with pSS and SLE [
Due to the limited supply of fresh exocrine gland tissues, this study focused on the circulating NK cells in the peripheral blood of pSS patients. Both CD56bright NK cells and CD56dim NK cells were found to be depleted within the peripheral blood compartment of pSS patients compared to healthy controls, while the latter decreased more. We also found that among NK cells, the percentage of CD56bright NK cells was increased and of CD56dim NK cells was decreased, resulting from the greater loss of CD56dim NK cell subset. Further analysis showed that the ratio of CD56bright NK cells relative to the total NK population to CD56dim NK cells was significantly elevated. Taken together, these results imply an imbalance of CD56 NK cell subsets in pSS, and this imbalance may reflect the immune status to some extent in pSS patients.
It is commonly recognized that serum IgG level, complement C3 and C4 consumption, and high ESSDAI are usually used to evaluate the disease activity in pSS patients. Goules et al. found that CD56+ NK cell incidence in the minor salivary glands of pSS patients was slightly increased, but not associated with the grade of inflammation [
In consistent with the previous report, our data showed that the proportion and absolute number of circulating CD3-CD56+ NK cells were significantly reduced in pSS patients versus controls [
There exist some limitations of this study. It is commonly recognized that CD56+ NK cell subsets in the salivary gland of pSS patients can be more suitable to reflect the situation in the gland. In this study, we mainly focused on the circulating CD56+ NK cell subsets. It is difficult to distinguish the CD56bright and CD56dim NK cell subsets by immunohistochemistry staining; also, the biopsy gland tissues of pSS patients are not enough to analyze with flow cytometry. In addition, the number of SLE patients was relatively small. We only included newly diagnosed SLE patients as controls, which limited the number of included patients.
In summary, our results suggest that the ratio of peripheral blood CD56bright NK to CD56dim NK has the potential to identify the pSS patients and was associated with serum IgG levels and complement C3 and C4 consumption. Further studies are required to better understand the crucial role of CD56 NK cell subsets in the pathogenesis of pSS in disease-affected tissues.
The data that support the findings of this study are available. If it is necessary, we will provide it at all.
The authors declare no conflict of interest.
Bingxia Ming and Tong Wu contributed equally to this work.
This work was supported by grants from the National Natural Scientific Foundation of China (No. 81771754 and No. 81901586) and the Tongji Hospital Clinical Research Flagship Program (No. 2019CR206).
Figure 1: comparison of function of peripheral NK cells and CD56 NK cell subsets between pSS patients and HC. (A) The percentage of CD107a+ cells in NK cells or CD56 NK cell subsets. Cells were collected from HCs (