ABSTRACT

Objective:

One-third of patients with primary Sjögren’s syndrome (PSS) do not have anti-SSA or -SSB antibodies. The risk of developing B-cell non-Hodgkin lymphoma (NHL) in PSS is significantly increased, and its prevalence is approximately 5%. There is a continuing need for new markers that can have diagnostic value in PSS and predict lymphoma development. In this study, we aimed to investigate the usability of netrin-1 as a diagnostic marker in PSS and its relationship with disease activity and predictors of lymphoma.

Methods:

Fifty-two PSS patients and 62 healthy volunteers were included in the study. The European Alliance of Associations for Rheumatology Sjögren’s syndrome disease activity index (ESSDAI) was used to evaluate systemic disease activity in PSS patients. Netrin-1 values were calculated by the quantitative sandwich enzyme immunoassay method using an ELISA kit (catalog number: E-EL-H2328; Elabscience, lot number: GZWTKZ5SWK, Texas, USA).

Results:

Serum netrin-1 levels were similar in PSS [90.3 (43.87-166.41)] and healthy controls [111.6 (63.39-171.36)] (p=0.190). Netrin-1 serum levels were associated only with lymphopenia (p=0.014), one of the predictive markers of lymphoma in PSS, but not with other markers (p>0.05) and ESSDAI score (r=0.637, p=0.067).

Conclusion:

Serum netrin-1 levels are not high in PSS and there is no significant correlation between netrin-1 and lymphoma predictive values, except lymphopenia, and the ESSDAI score, which is an indicator of disease activity.

Introduction

Primary Sjögren’s syndrome (PSS) is a chronic autoimmune disease not related to other autoimmune diseases, characterized by lymphocytic infiltration of exocrine glands, mainly with glandular dysfunction of salivary and lacrimal glands. Keratoconjunctiva sicca (dry eyes) and xerostomia (dry mouth) are the main symptoms.^[1] Since two-thirds of PSS patients are positive for anti-SSA/Ro or -SSB/La autoantibodies, these antibodies are considered one of the main criteria in the 2016 American College of Rheumatology (ACR)/European Alliance of Associations for Rheumatology (EULAR) PSS classification.^[2] Since anti-SSA or SSB antibodies are negative in one-third of PSS patients, many studies have been conducted to detect new autoantibodies that can be used in diagnosing PSS. However, the need for diagnostic biomarkers continues. These novel autoantibodies include autoantibodies against serum salivary gland protein-1, parotid secretion protein, muscarinic-3 receptor, and carbonic anhydrase-6.^[3-5]

B-cell non-Hodgkin lymphoma occurs in approximately 5% of patients with PSS and is considered to be the main source of mortality.^[6] Studies have been ongoing for a long time to determine the markers (laboratory, pathological and clinical) that may have predictive value in developing lymphoma in PSS. Among the best markers with prognostic value in the development of lymphoma are mixed cryoglobulinemia, cryoglobulinemic vasculitis, and persistent salivary gland swelling.^[7-17] Other lymphoma predictive values include skin purpura which may be associated with cryoglobulinemia, low C4 level and organ involvement associated with cryoglobulinemic vasculitis (glomerulonephritis, peripheral neuropathy);^[18,19] high MALT involvement in salivary gland histopathology; the presence of monoclonal gammopathy; RF positivity;^[20-22] lymphopenia; neutropenia; increased serum beta-2 microglobulin; elevated free immunoglobulin light chains; splenomegaly; lymphadenopathy; cytokines; chemokines; growth factors; monoclonal B lymphocyte expansion in metachronous tissue histopathology; genetic abnormalities and EULAR Sjögren’s syndrome disease activity index (ESSDAI).^[23]

The need for new markers associated with diagnosis, disease activity indicators, and lymphoma development in PSS continues. The relationship of netrin-1 with PSS has yet to be evaluated. In this study, we investigated the value of netrin-1 molecule as a diagnostic marker and disease activity indicator in PSS and the relationship between serum levels and predictors of lymphoma. Netrin-1 is a laminin-like matrix protein from the axonal guide protein family. Netrin-1 acts as a chemorepulsan and inhibits the migration of neutrophils, monocytes, and lymphocytes through Unc5b and adenosine A2B receptors.^[24-26] Netrin-1 plays a pathogenic role in obesity, the development of atherosclerosis,^[27,28] the initiation of sepsis and inflammation,^[24] osteoporosis by stimulating osteoclast differentiation,^[29] and the development of inflammatory arthritis.^[30] In our previous study, we showed that netrin-1 was found to be higher in the serum of patients with systemic sclerosis (SSc) compared to healthy controls (p<0.0001) and that it may be associated with the pathogenesis of systemic sclerosis.^[31]. Deleted in Colorectal Carcinoma (DCC) is a transmembrane netrin-1 receptor that actively induces cell death when dissociated from the netrin-1 ligand. When netrin-1 binds to the DCC receptor, it causes inhibition of DCC-induced apoptosis. It contributes to the increase in the population of diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) tumor cells.^[32] In addition, netrin-1 causes progression in plasma cell malignancies.^[33] These associations of netrin-1 with lymphoid malignancies prompted us to investigate the relationship of netrin-1 with markers that can predict lymphoma in PSS.

Materials and Methods

Study Design

This study was designed as an analytical case-control study. Fifty-two patients (47 women, five men) followed in the rheumatology department of Ankara City Hospital and classified according to the 2016 ACR/EULAR criteria^[34] for PSS were included in the PSS group. In the control group, 62 healthy volunteers (55 females, seven males) of similar age and gender to the PSS group were selected. Active infection, pregnancy, malignant disease, and the presence of rheumatological disease other than PSS were the exclusion criteria. Superficial and abdominal ultrasonography (USG) for lymphadenopathy and splenomegaly; pulmonary function test, carbon monoxide diffusion test or high-resolution tomography for interstitial lung disease; neurological examination and electromyography for peripheral neurologic involvement; urine microscopy and renal biopsy histopathology for glomerulonephritis; parotid gland USG for parotid gland swelling; joint examination and joint USG for arthritis; skin examination for purpura was used to detect lymphoma predictors in the PSS group. The ESSDAI score was used to evaluate systemic disease activity in PSS.^[35] To calculate this index score, a total of 12 areas, 11 areas related to organ involvement and one biological place reflecting B-cell activity, were examined, and scores were given to the patients. According to the ESSDAI score, patients were divided into groups defining systemic disease activity as low (5>), moderate (>5 and <14), or severe (>14). Individuals with hypertension, diabetes mellitus, chronic heart disease, or chronic lung disease were included in the study groups in minimum numbers, and individuals with any of these diseases were considered positive for the presence of comorbid disease. Informed consent was obtained from all individuals included in the study before the study. Dates are given in DD/MM/YYYY format.

Obtaining Serum Samples and Determination of Serum Netrin-1 Level

Venous blood samples in 10 mL vacuum tubes were centrifuged at 1300 x g for an average of 10 minutes. Serum samples were stored in eppendorf tubes at -80 °C until analysis. Serum netrin-1 levels were measured using a quantitative immunoassay method with the catalog number E-EL-H2328 from elabscience, lot number GZWTKZ5SWK, in Texas, USA. Before adding the specific detection antibody rich in biotin for netrin-1, serum samples and standards were incubated with their specific antibodies at 37 °C for approximately 1.5 hours after addition to the micro-ELISA plate wells. Biotin-rich human netrin-1-specific detection antibodies and Avidin-Horseradish Peroxidase (HRP) were then added, and the samples were then incubated at 37 °C for 30 minutes. After the free components were separated by washing, the substrate solution was added to all wells. After this process was completed, a blue color was observed only in the Avidin-HRP conjugate, human netrin-1 wells and biotin-rich detection antibodies. The enzyme-substrate reaction ended after the addition of the stopping solution, resulting in a yellow color in the reaction. A spectrophotometric microplate reader with a wavelength of 450 nm was used to detect optical density levels, which is considered an indirect indicator of Human netrin-1 concentrations. Serum levels of human netrin-1 were calculated using optical density standard curves. The sensitivity used for netrin-1 at levels in the range of 31.25 to 2.000 pg/mL was determined. Inter- and intra-assay precision of <10% was obtained for all levels of netrin-1 concentrations.

Statistical Analysis

The Statistical Packages for the Social Sciences (SPSS) version 22.0 package program was used for the evaluation of statistical analyzes, and the p<0.05 level was considered statistically significant. Normality distribution fit in continuous variables was determined using analytical methods such as Kolmogorov-Smirnov/Shapiro-Wilk tests and histogram/probability graphs. Descriptive statistical results were shown as mean and standard deviation for normally distributed variables, and median [interquartile range, (25%-75%)] for non-normally distributed variables. The Independent Samples t-test and Mann-Whitney U test were used to evaluate the statistical significance difference in pairwise comparisons. Spearman correlation test was used to evaluate the correlation analysis between continuous variables. One-Way ANOVA Post-hoc Tukey test was used to evaluate variables with normal distribution, and Independent Samples Kruskal-Wallis test was used to evaluate variables that did not show normal distribution. Bonferroni correction was performed before the One-Way ANOVA Post-hoc Tukey test and the Independent Samples Kruskal-Wallis test. The Fisher’s exact tests and chi-square test were used to compare categorical data.

Results

This study was designed as an analytical case control study. The PSS group consisting of 52 patients with a mean age of 49.08±7.23 and a healthy control group consisting of 62 patients with a mean age of 51.06±9.41 were included in the study. Age, gender, body mass index, smoking and presence of comorbid disease in the control group were similar to the PSS group (p<0.05). No significant difference was found between PSS and control groups in comparison of median values for netrin-1 [90.3 (43.87-166.41), 111.6 (63.39-171.36), p=0.190, respectively]. Demographic characteristics and laboratory parameters of PSS and control groups are shown in Table 1.

Table 1

There was no significant difference in median values of netrin-1 between patients with (n=4) and those without (n=48) interstitial lung disease (n=48) in the PPS group [respectively, 115.17 (35.3-395.2), 90.3 (47.2-162.5), p=0.882]. In PSS patients, the median values of netrin-1 were found to be similar between those with negative antibody (anti-SSA/RO52 and -SSB) (n=11) and those with any antibody positivity (anti-SSA/RO52 or -SSB) (n=41) [respectively, 90.54 (38.04-184.4), 90.06 (47.3-159.3), p=0.920].

In the multiple comparisons made in terms of netrin-1 levels between groups receiving different types of treatment in PSS, netrin-1 levels were found to be similar between groups receiving other types of treatment (p=0.342). There was no significant difference in netrin-1 levels between those who received any treatment for PSS and those who did not receive the same treatment (p>0.05). The comparison of netrin-1 levels according to medical treatment subtypes in the PSS group is shown in Table 2.

Table 2

While netrin-1 serum levels are associated only with presence of lymphopenia [yes=159.34 (91.2-213.8), no=81.29 (42.3-147.5) p=0.014], which is a predictive marker of lymphoma in PSS, it is not correlated with other features (p>0.05). The relationship between the factors predicting lymphoma and netrin-1 levels in PSS is shown in Table 3.

Table 3

While there was a significant correlation between netrin-1 and spot urine protein amount (r=0.278, p=0.046) and lymphocyte level (r=-0.343, p=0.013), no significant correlation was found between other study parameters (p>0.05) (Table 4).

Table 4

Discussion

The absence of anti-SSA or anti-SSB antibodies in one-third of PSS patients and the lack of markers that can be used in disease activation and lymphoma prediction have revealed the necessity of investigating new autoantibodies associated with PSS. It has been previously reported that netrin-1 may be related to different mechanisms in the pathogenesis of some lymphomas,^[32] multiple myeloma,^[33] solid tumors,^[36] and SSc,^[31] a connective tissue disease. In this study, we investigated the utility of netrin-1 as an indicator of disease activity in PSS and its relationship with known predictors of lymphoma. Our results showed that netrin-1 levels were not high in the serum of PSS patients, and there was no significant correlation between disease activity and ESSDAI score. In addition, this study showed a substantial relationship between netrin-1 levels and lymphopenia, one of the known predictors of lymphoma in PSS. Still, no critical relationship exists between other predictors.

There are a limited number of studies investigating the relationship of netrin-1 with rheumatological diseases. In two of these studies, the relationship between netrin-1 and antibodies in rheumatoid arthritis (RA) synovial tissues,^[30,37] while in other studies, netrin-1 levels were investigated in the serum of patients with familial Mediterranean fever (FMF)^[38] and SSc^[31] patients. It was determined that there was a significant decrease in inflammation and joint erosion in the group treated with anti-netrin-1/anti-Unc5b monoclonal antibody injection (n=6) compared to the untreated group (n=10) from 8-week-old arthritis mice (p<0.001).^[30] Unlike this study, Schubert et al.^[37] reported that the expression of UNC5B (4-fold) and UNC5C (769-fold) from netrin-1 receptors was higher in RA (n=5) and osteoarthritis (OA) (n=6) synovial tissues compared to healthy subjects (n=3) synovial tissues. They also reported that treatment of RA and OA synovial tissues with netrin-1 results in the inhibition of the migration of synovial fibroblasts, and thus netrin-1 could reduce cartilage degeneration. In a study where we evaluated serum netrin-1 levels between FMF patients (n=42) and healthy controls (n=44), we found netrin-1 levels to be similar between the two groups (p=0.19).^[38] In another study, we evaluated netrin-1 levels in the sera of SSc patients (n=56) compared to healthy controls (n=58). In this study, we found that netrin-1 levels were significantly higher in the sera of SSc patients (p<0.0001).^[31] Studies supporting the possible contribution of netrin-1 to the pathogenesis of SSc showed that profibrotic cytokine and extracellular matrix protein synthesis in SSc were induced by M2 macrophages^[39,40] and netrin-1 increased the expression of M2 macrophage markers.^[41-43] It has also been reported that netrin-1 promotes the development of fibrosis in human SSc lung cell culture and bleomycin-induced mouse lung.^[44,45] In this study, we compared serum netrin-1 levels between PSS patients (n=52) and healthy controls (n=62), we found netrin-1 levels to be similar between the groups, and we did not find a significant correlation between netrin-1 and the disease activation indicator ESSDAI score (p>0.05). Although the data on the relationship of netrin-1 with rheumatological diseases come mostly from small-scale or cross-sectional studies, further randomized controlled studies are needed to better understand its role in rheumatological diseases.

A study by Broutier et al.^[32] using a transgenic mouse model showed that netrin-1 contributes to the development of DLBCL and MCL by inhibiting apoptosis on DCC receptors. In addition, this study determined that there was a decrease in tumor cell density and an increase in DNA fragmentation in tumor cell lines with antibodies blocking netrin-1. Nagoshi et al.^[33] showed that transcriptional dysregulation of the netrin-1 receptor DCC in multiple myeloma cell lines plays a role in the progression of plasma cell malignancy. Netrin-1 has been found to cause the inhibition of p53-related apoptosis by stabilizing inactive p53 expression through its receptors.^[46,47] It has been reported to be associated with hepatocellular, breast, lung, colorectal, and pancreatic cancers.^[34] These associations of netrin-1 with lymphoid and solid malignant diseases prompted us to investigate its relationship with lymphoma predictors in PSS. In our study, a significant relationship was found between netrin-1 and only lymphopenia, but no significant relationship was found between other lymphoma predictive factors.

Study Limitations

The limitations of this study are that it is a cross-sectional study, there is an insufficient number of subjects in some groups that predict lymphoma, and ectopic germinal-like structures and focus scores were not evaluated in salivary gland biopsy. In our study, although the median values of netrin-1 were higher in some lymphoma predictor subgroups (hypergammaglobulinemia, lymphadenopathy, splenomegaly, ESSDAI, parotid gland enlargement, monoclonal gammopathy, glomerulonephritis, leukopenia, neutropenia), no statistically significant results could be obtained. This may be because the statistical results are affected due to the small number of subjects in the subgroups. Although we did not find a significant relationship between netrin-1 and most of the variables that predict lymphoma in PSS in this study, the close relationship of netrin-1 with lymphoproliferative and solid malignancies makes it necessary to investigate its role in the development of lymphoma in PSS with further prospective studies.

Conclusion

Ethics

Ethics Committee Approval: The study was approved by the ethics committee of the University where the study was conducted (Ankara City Hospital Ethics Committee - approval number: 460; date: 30.09.2020).

Informed Consent: Informed consent was obtained from all individuals included in the study before the study.

Peer-review: Externally peer-reviewed.

Authorship Contributions

Concept: A.K., H.E.K., E.F.O., Y.M., E.A., K.O., Ö.E., Ş.E., Design: A.K., H.E.K., E.F.O., Y.M., E.A., K.O., Ö.E., Ş.E., Data Collection or Processing: A.K., H.E.K., E.F.O., Y.M., E.A., K.O., Ö.E., Ş.E., Analysis or Interpretation: A.K., H.E.K., E.F.O., Y.M., E.A., K.O., Ö.E., Ş.E., Literature Search: A.K., H.E.K., E.F.O., Y.M., E.A., K.O., Ö.E., Ş.E., Writing: A.K., H.E.K., E.F.O., Y.M., E.A., K.O., Ö.E., Ş.E.

Conflict of Interest: No conflict of interest was declared by the author.

Financial Disclosure: The author declare that they have no relevant financial disclosures.

References

1Du AX, Gniadecki R, Osman M. Biomarkers of B cell activation in autoimmune connective tissue diseases: More than markers of disease activity. Clin Biochem 2022;100:1-12.

2Mielle J, Tison A, Cornec D, Le Pottier L, Daien C, Pers JO. B cells in Sjögren’s syndrome: from pathophysiology to therapeutic target. Rheumatology (Oxford) 2012;60:2545-60.

3Shen L, Suresh L, Lindemann M, et al. Novel autoantibodies in Sjögren’s syndrome. Clin Immunol 2012;145:251-5.

4Chen Y, Zheng J, Huang Q, et al. Autoantibodies against the second extracellular loop of M3R Do neither induce nor indicate primary Sjogren’s syndrome. PLoS One 2016;11:e0149485.

5Theander E, Jonsson R, Sjöström B, Brokstad K, Olsson P, Henriksson G. Prediction of Sjögren’s Syndrome Years Before Diagnosis and Identification of Patients With Early Onset and Severe Disease Course by Autoantibody Profiling. Arthritis Rheumatol 2015;67:2427-36.

6Goules AV, Tzioufas AG. Lymphomagenesis in Sjögren’s syndrome: predictive biomarkers towards precision medicine. Autoimmun Rev 2019;18:137-43.

7De Vita S, De Marchi G, Sacco S, Gremese E, Fabris M, Ferraccioli G. Preliminary classification of nonmalignant B cell proliferation in Sjögren’s syndrome: perspectives on pathobiology and treatment based on an integrated clinico-pathologic and molecular study approach. Blood Cells Mol Dis 2001;27:757-66.

8De Vita S, Gandolfo S, Zandonella Callegher S, Zabotti A, Quartuccio L. The evaluation of disease activity in Sjögren’s syndrome based on the degree of MALT involvement: glandular swelling and cryoglobulinaemia compared to ESSDAI in a cohort study. Clin Exp Rheumatol 2018;36:150-6.

9Baimpa E, Dahabreh IJ, Voulgarelis M, Moutsopoulos HM. Hematologic manifestations and predictors of lymphoma development in primary Sjögren syndrome: clinical and pathophysiologic aspects. Medicine (Baltimore) 2009;88:284-93.

10Quartuccio L, Isola M, Baldini C, et al. Biomarkers of lymphoma in Sjögren’s syndrome and evaluation of the lymphoma risk in prelymphomatous conditions: results of a multicenter study. J Autoimmun 2014;51:75-80.

11Brito-Zerón P, Kostov B, Fraile G, et al. Characterization and risk estimate of cancer in patients with primary Sjögren syndrome. J Hematol Oncol 2017;10:90.

12Papageorgiou A, Ziogas DC, Mavragani CP, et al. Predicting the outcome of Sjogren’s syndrome-associated non-hodgkin’s lymphoma patients. PloS One 2015;10:e0116189.

13Fragkioudaki S, Mavragani CP, Moutsopoulos HM. Predicting the risk for lymphoma development in Sjogren syndrome: an easy tool for clinical use. Medicine (Baltimore) 2016;95:e3766.

14Tzioufas AG, Boumba DS, Skopouli FN, Moutsopoulos HM. Mixed monoclonal cryoglobulinemia and monoclonal rheumatoid factor cross-reactive idiotypes as predictive factors for the development of lymphoma in primary Sjögren’s syndrome. Arthritis Rheum 1996;39:767-72.

15Theander E, Vasaitis L, Baecklund E, et al. Lymphoid organisation in labial salivary gland biopsies is a possible predictor for the development of malignant lymphoma in primary Sjögren’s syndrome. Ann Rheum Dis 2011;70:1363-8.

16Retamozo S, Gheitasi H, Quartuccio L, et al. Cryoglobulinaemic vasculitis at diagnosis predicts mortality in primary Sjögren syndrome: analysis of 515 patients. Rheumatology (Oxford) 2016;55:1443-51.

17De Vita S, Quartuccio L, Salvin S, Corazza L, Zabotti A, Fabris M. Cryoglobulinaemia related to Sjogren’s syndrome or HCV infection: differences based on the pattern of bone marrow involvement, lymphoma evolution and laboratory tests after parotidectomy. Rheumatology (Oxford) 2012;51:627-33.

18Visser A, Doorenspleet ME, de Vries N, et al. Acquisition of N-glycosylation sites in immunoglobulin heavy chain genes during local expansion in parotid salivary glands of primary sjögren patients. Front Immunol 2018;9:491.

19De Vita S, Gandolfo S. Predicting lymphoma development in patients with Sjögren’s syndrome. Expert Rev Clin Immunol 2019;15:929-38.

20Tzioufas AG, Kapsogeorgou EK, Moutsopoulos HM. Pathogenesis of Sjögren’s syndrome: what we know and what we should learn. J Autoimmun 2012;39:4-8.

21Moutsopoulos HM. Sjögren’s syndrome: autoimmune epithelitis. Clin Immunol Immunopathol 1994;72:162-5.

22Teos LY, Alevizos I. Genetics of Sjögren’s syndrome. Clin Immunol 2017;182:41-7.

23Retamozo S, Brito-Zerón P, Ramos-Casals M. Prognostic markers of lymphoma development in primary Sjögren syndrome. Lupus 2019;28:923-36.

24Ly NP, Komatsuzaki K, Fraser IP, et al. Netrin-1 inhibits leukocyte migration in vitro and in vivo. Proc Natl Acad Sci USA 2005;102:14729-34.

25Mirakaj V, Thix CA, Laucher S, et al. Netrin-1 dampens pulmonary inflammation during acute lung injury. Am J Respir Crit Care Med 2010;181:815-24.

26Rosenberger P, Schwab JM, Mirakaj V, et al. Hypoxia-inducible factor-dependent induction of netrin-1 dampens inflammation caused by hypoxia. Nat. Immunol 2009;10:195-202.

27VanGils JM, Derby MC, Fernandes LR, et al. The neuroimmune guidance cue netrin-1 promotes atherosclerosis by inhibiting the emigration of macrophages from plaques. Nat Immunol 2012;13:136-43.

28Ramkhelawon B, Hennessy EJ, Ménager M, et al. Netrin-1 promotes adipose tissue macrophage retention and insulin resistance in obesity. Nat Med 2014;20:377-84.

29Mediero A, Ramkhelawon B, Perez-Aso M, Moore KJ, Cronstein BN. Netrin-1 is a critical autocrine/paracrine factor for osteoclast differentiation. J Bone Miner Res 2014;30:837-54.

30Mediero A, Wilder T, Ramkhelawon B, Moore KJ, Cronstein BN. Netrin-1 and its receptor Unc5b are novel targets for the treatment of inflammatory arthritis. FASEB J 2016;30:3835-44.

31Maraş Y, Kor A, Oğuz EF, Sarı A, Gök K, Akdoğan A. Serum netrin-1 levels in systemic sclerosis patients with capillary abnormalities. Egypt Rheumatol 2023;45:51-4.

32Broutier L, Creveaux M, Vial J, et al. Targeting netrin-1/DCC interaction in diffuse large B-cell and mantle cell lymphomas. EMBO Mol Med 2016;8:96-104.

33Nagoshi H, Taki T, Chinen Y, et al. Transcriptional dysregulation of the deleted in colorectal carcinoma gene in multiple myeloma and monoclonal gammopathy of undetermined significance. Genes Chromosomes Cancer 2015;54:788-95.

34Shiboski CH, Shiboski SC, Seror R, et al. 2016 American College of Rheumatology/European League Against Rheumatism classification criteria for primary Sjögren’s syndrome: a consensus and data-driven methodology involving three international patient cohorts. Arthritis Rheumatol 2017;69:35-45.

35Seror R, Ravaud P, Bowman SJ, et al. EULAR Sjogren’s syndrome disease activity index: development of a consensus systemic disease activity index for primary Sjogren’s syndrome. Ann Rheum Dis 2010;69:1103-9.

36Kefeli U, Ucuncu Kefeli A, Cabuk D, et al. Netrin-1 in cancer: Potential biomarker and therapeutic target? Tumour Biol 2017;39:1010428317698388.

37Schubert T, Denk A, Mägdefrau U, et al. Role of the netrin system of repellent factors on synovial fibroblasts in rheumatoid arthritis and osteoarthritis. Int J Immunopathol Pharmacol 2009;22:715-22.

38Atalar E, Gök K, Oğuz EF, Kor A, Maraş Y. Plasma netrin-1 levels in Familial Mediterranean fever: a potential biomarker? J Exp Clin Med 2022;39:1202-6.

39Bhattacharya S, Aggarwal A. M2 macrophages and their role in rheumatic diseases. Rheumatol Int 2019;39:769-80.

40Funes SC, Rios M, Escobar-Vera J, Kalergis AM. Implications of macrophage polarization in autoimmunity. Immunology 2018;154:186-95.

41Ranganathan P, Mohamed R, Jayakumar C, Ramesh G. Guidance cue netrin-1 and the regulation of inflammation in acute and chronic kidney disease. Mediators Inflamm 2104;2014:525891.

42Zhang Y, Chen P, Di G, Qi X, Zhou Q, Gao H. Netrin-1 promotes diabetic corneal wound healing through molecular mechanisms mediated via the adenosine 2B receptor. Sci Rep 2018;8:5994.

43Ranganathan PV, Jayakumar C, Ramesh G. Netrin-1-treated macrophages protect the kidney against ischemia-reperfusion injury and suppress inflammation by inducing M2 polarization. Am J Physiol Renal Physiol 2013;304:948-57.

44Gao R, Peng X, Perry C, et al. Macrophage-derived netrin-1 drives adrenergic nerve-associated lung fibrosis. J Clin Invest 2021;131:e136542.

45Sun H, Zhu Y, Pan H, et al. Netrin-1 Regulates Fibrocyte Accumulation in the Decellularized Fibrotic Sclerodermatous Lung Microenvironment and bleomycin-Induced Pulmonary Fibrosis. Arthritis Rheumatol 2016;68:1251-61.

46Mehlen P, Guenebeaud C. Netrin-1 and its dependence receptors as original targets for cancer therapy. Curr Opin Oncol 2010;22:46-54.

47Tanikawa C, Matsuda K, Fukuda S, Nakamura Y, Arakawa H. p53RDL1 regulates p53-dependent apoptosis. Nat Cell Biol 2003;5:216-23.

Evaluation of the relationship between lymphoma predictors, disease activity, and netrin-1 in primary Sjögren’s syndrome