Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) is a type I transmembrane protein belonging to the TNFR superfamily. After activated by its ligand GITRL, GITR could influence the activity of effector and regulatory T cells, participating in the development of several autoimmune and inflammatory diseases included rheumatoid arthritis and autoimmune thyroid disease. We previously reported that serum GITRL levels are increased in systemic lupus erythematosus (SLE) patients compared with healthy controls (HC). Here, we tested serum soluble GITR (sGITR) and GITRL levels in 41 primary Sjögren’s syndrome (pSS) patients and 29 HC by ELISA and correlated sGITR and GITRL levels with clinical and laboratory variables. GITR and GITRL expression in labial salivary glands was detected by immunohistochemistry. pSS patients had significantly increased serum levels of sGITR and GITRL compared with controls (GITR: 5.66 ± 3.56 ng/mL versus 0.50 ± 0.31 ng/mL;
Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) is a type I transmembrane protein belonging to the TNFR superfamily, which was originally discovered by Nocentini et al. as a gene upregulated in dexamethasone-treated murine T cell hybridomas [
Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease characterized by keratoconjunctivitis sicca, xerostomia, and extraglandular abnormalities [
The study group comprised 41 patients (39 women and 2 men) with a mean age of
All patients underwent extensive medical examinations and serological evaluations, including measurements of antinuclear antibodies (ANA), anti-Ro/SSA antibody (A-SSA), anti-La/SSB antibody (A-SSB), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA), and rheumatoid factor (RF). In addition, the numbers of cellular infiltration per 4 mm2 of tissue (focus index, FI) were measured. The clinical data from the patients were recorded in Table
Characteristics of pSS patients and the control subjects.
pSS |
Control |
|
---|---|---|
Age (years) |
|
|
Sex (female/male) | 39/2 | 28/1 |
Disease duration (months) |
|
— |
Arthritis (%) | 6 (14.63) | — |
Fever (%) | 8 (19.51) | — |
Anemia (%) | 12 (29.27) | — |
Leukopenia (%) | 20 (48.78) | — |
Thrombocytopenia (%) | 4 (9.76) | — |
Renal disease (%) | 7 (17.07) | — |
Pulmonary interstitial changes (%) | 5 (12.20) | — |
Autoimmune liver disfunction (%) | 4 (9.76) | — |
ANA positive (%) | 36 (87.80) | — |
A—SSA positive (%) | 34 (82.93) | — |
A—SSB positive (%) | 19 (46.34) | — |
ESR (mm/H) |
|
— |
CRP (mg/L) |
|
— |
IgG (g/L) |
|
— |
IgM (g/L) |
|
— |
IgA (g/L) |
|
— |
RF (IU/L) |
|
— |
Focus index (1–3) |
|
— |
pSS: primary Sjögren’s syndrome; ANA: antinuclear antibody; A-SSA: anti-Ro/SSA antibody; A-SSB: anti-La/SSB antibody; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein; IgG: immunoglobulin G; IgM: immunoglobulin M; IgA: immunoglobulin A; RF: rheumatoid factor; Focus index: the number of foci per 4 mm2 of tissue.
Values are expressed as mean ± standard deviation.
A volume of 5 mL peripheral venous blood was collected from each patient and health control subject and waited to clot at room temperature for 2 hours. Samples were then centrifuged for 10 minutes at 800 g. The serum samples were separated and frozen at −80°C until further analysis.
LSG biopsies were taken from patients with sicca symptoms. For this, local anesthetic was injected into the lower lip and a small incision to the right or left of the lip midline was made. Four or five LSG lobules were harvested and placed into Carnoy’s fixative for 24 hours. Standard paraffin preparations were prepared for sectioning at a thickness of 3
Serum sGITR and GITRL levels were measured using ELISA kits (RayBiotech Inc.) according to the manufacturer’s protocols. Briefly, serum samples (1 : 50 dilution) and standards were added to the 96-well plate and incubated overnight at 4°C. After incubation for 2 hours and washing 3 times, biotinylated antihuman GITRL antibodies were added, followed by incubation with HRP-conjugated streptavidin and color development with TMB substrate solution. The intensity of the color reaction was measured by a microplate reader (Bio-Rad, Beijing, China) at a wavelength of 450 nm. Concentrations of sGITR and GITRL were determined by a standard curve according to the manufacturer’s instructions.
The fixed LSG biopsy specimen slides were fixed in Carnoy’s fixative and embedded in paraffin wax. Paraffinized LSG tissues were sectioned to 3
Data were presented as mean ± standard deviation unless specified otherwise. Statistical analysis was performed using SPSS for Windows (version 11.5). The Mann-Whitney rank sum test or Kruskal-Wallis tests were used to compare sGITR and GITRL levels. The correlation between GITRL/sGITR levels and various values were analyzed by Spearman’s rank correlation coefficient. A value of
Serum GITRL and sGITR levels in pSS (
Comparison of serum GITRL and sGITR levels between pSS and HC. (a) Serum GITRL levels were significantly elevated in SS patients versus HC. (b) Serum sGITR levels were also significantly elevated in SS patients versus HC. Each symbol represents an individual patient and healthy donor. Horizontal lines indicate median values. GITRL: glucocorticoid-induced TNFR-related protein ligand; sGITR: soluble glucocorticoid-induced TNFR-related gene; SS: Sjögren’s syndrome; HC: healthy control.
We further divided pSS patients into groups with or without extraglandular manifestations according to their symptoms. pSS patients with sicca symptoms only such as dry mouth or dry eye were defined as the non-extraglandular manifestations group, while other patients with fever, arthritis, anemia, leukopenia, thrombocytopenia, renal disease, pulmonary interstitial changes, or autoimmune liver dysfunction were extraglandular manifestations group. As shown in Figure
Comparison of serum GITRL and sGITR levels between pSS patients with or without extra-glandular manifestations. (a) Serum GITRL levels exhibited elevation in pSS patients with extra-glandular manifestations (
The FI was recorded as the number of foci per 4 mm2 of LSGs. We divided pSS patients into various groups according to their FI. As shown in Figure
Comparison of serum GITRL and sGITR levels among pSS patients with different foci index. pSS patients were divided according to the foci index (1~3) in labial salivary glands (LSGs) of lymphocytic infiltration (see Methods). (a) Serum GITRL levels were significantly higher in pSS patients with serious sialadenitis. (b) Serum sGITR levels were also elevated as foci index increased in pSS patients.
To further determine the relationship between serum GITRL/sGITR levels and laboratory test results including the titers of ANA, A-SSA, A-SSB, ESR, CRP, RF, and Ig levels, it was found that both serum GITRL and sGITR levels were positively correlated with ESR (GITRL:
Comparison of serum GITRL and sGITR levels between pSS patients with normal or abnormal laboratory values.
Parameter | GITRL (ng/mL) | sGITR (ng/mL) | ||||
---|---|---|---|---|---|---|
Normal | Abnormal |
|
Normal | Abnormal |
| |
Mean ± SD |
Mean ± SD |
Mean ± SD |
Mean ± SD |
|||
A—SSA |
|
|
0.1362 |
|
|
0.1657 |
A—SSB |
|
|
0.5830 |
|
|
0.8857 |
ANA |
|
|
**0.0050 |
|
|
*0.0114 |
CRP |
|
|
**0.0078 |
|
|
*0.0231 |
RF |
|
|
**0.0050 |
|
|
**0.0075 |
IgM |
|
|
*0.0431 |
|
|
0.2428 |
IgA |
|
|
**0.0069 |
|
|
*0.0381 |
WBC |
|
|
*0.0158 |
|
|
*0.0110 |
Laboratory values such as A-SSA, A-SSB, ANA, and RF positive were defined as abnormal, while laboratory parameters such as CRP, IgM, and IgA above limit values were defined as abnormal. In addition, WBC abnormal means leukopenia.
A-SSA: anti-Ro/SSA antibody; A-SSB: anti-La/SSB antibody; ANA: antinuclear antibody; CRP: C-reactive protein; RF: rheumatoid factor; IgM: immunoglobulin M; IgA: immunoglobulin A; WBC: white blood count.
Correlation of serum GITRL and sGITR levels with laboratory values. (a), (b) Positive correlation was observed between serum GITRL levels and ESR, IgG. (c), (d) Positive correlation was also seen between serum sGITR levels and ESR, IgG. ESR: erythrocyte sedimentation rate; IgG: immunoglobulin G.
To evaluate the local effect of GITRL and GITR in LSGs, we applied immunohistochemical staining to determine the expression of GITRL and GITR in LSGs. The freshly explanted lower lip biopsy specimens were sectioned and stained with anti-GITRL and anti-GITR antibodies. All 10 pSS samples exhibited distinct expression of GITRL, while 6 of the 10 samples were positive for GITR. In contrast, none of the LSGs from the sicca complainers exhibited GITRL or GITR expression. Six specimens from pSS patients stained for GITR showed similar pattern of GITRL and GITR expression; that is, GITRL was prominent in infiltrating lymphocytes and ductal cells, while GITR was mainly expressed in infiltrating lymphocytes with a weak expression observed on ductal cells. Overall, the expression of GITRL was stronger and more widely distributed than GITR. Both GITRL and GITR were expressed on lymphocytic infiltrates and to a lesser degree in the acinar components (Figure
High expression of GITRL and GITR in labial salivary glands from patients with pSS. The labial salivary glands (LSG) of pSS patients exhibit increased GITRL and GITR expression. Shown is the expression of the GITRL and GITR in the labial salivary glands of sicca complainer (
In this study, we determined the serum levels of sGITR and GITRL in pSS patients and further revealed the positive correlation of their expression levels with IgG and ESR, which were in association with disease severity of pSS. In addition, we have shown for the first time the expression pattern of GITR and GITRL in the salivary glands of patients with pSS.
Several studies including our recent findings have suggested that GITR-GITRL system is involved in the pathogenesis of autoimmune disease including rheumatoid arthritis (RA) and SLE [
Recent studies have suggested that in most of inflammatory and autoimmune diseases, the activation of GITR-GITRL pathway promotes leucocyte extravasation, increases T lymphocyte activation, and partially reverses the immunosuppressive function of Treg [
In this study, we have demonstrated a close correlation of serum elevated GITRL and sGITR with the increased degree of lymphocytic infiltration in patients with pSS. Moreover, GITR and GITRL were readily detected in the lymphocytic foci and periductal areas of the LSGs. In contrast, the LSGs of healthy control subjects did not express GITR or GITRL. Moreover, we have further detected a sharp decrease of the stimulated salivary flow in the pSS patients with glandular cells positively stained for GITRL (data not shown). Our findings suggest that GITR and GITRL may play an important role in the sialadenitis suffered by patients with pSS. In addition, our investigations have revealed a close correlation of circulating sGITR and GITRL levels with the disease activity and severity in pSS patients. Our data have clearly shown that serum levels of sGITR and GITRL positively correlate with ESR and IgG, and exhibit elevation in the groups with extra-glandular manifestations as well as abnormal laboratory parameters such as ANA, CRP, IgM, IgA, and WBC. We provide new evidence indicating involvement of GITR/GITRL overactivation in the disease pathophysiology of pSS, which may serve as a new biomarker to assess the disease activity and severity of pSS.
Our findings indicate the possible involvement of GITR-GITRL pathway in the pathogenesis of pSS. Further investigations on the systemic and localized effects of GITR and GITRL in pSS may facilitate the development of targeting this molecule pathway for the treatment of pSS.
The authors declare that they have no financial and personal relationships with other people or organizations that can inappropriately influence their work, and there is no professional or other personal interest of any product, service and/or company that influence them.
This work was supported by the National Natural Science Foundation of China (NSFC no. 30701129, NSFC no. 30901332, NSFC no. 81172845, NSFC no. 81273294), the Natural Science Foundation of Jiangsu Province (no. BK2011851, no. BK2012875), the National “Eleven, Five” Science and Technology Support Program (no. 2008BAI59B03), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).