INTRODUCTION:

Systemic sclerosis is a systemic rheumatic autoimmune disease that involves multiple organs. The etiology of systemic sclerosis has not been certainly discovered. The pathogenesis of this disease is assumed to involve various factors including genetic, infectious, environmental, and drug factors. This disease has a different prognosis in each affected individual depending on the organs involved^1,2,3.

Systemic sclerosis has a wide distribution worldwide but the incidence is quite low. This disease is more frequently found in women than men, with a varied female/male ratio between 3:1 to 14:1. The prevalence of this disease is 13.5-44.3 cases per 100,000 population in North America and 7.2-33.9 cases per 100,000 population in Europe². Data on patients with systemic sclerosis in Indonesia remains unavailable. However, based on data from several hospitals, it is stated that there has been an increase in the number of visits of systemic sclerosis patients in several patient Rheumatology clinic. At the Rheumatology outpatient clinic of Hasan Sadikin General Hospital, Bandung, for example, the number of sclerosis systemic patients who actively controls to the Rheumatology clinic is the third after Systemic Lupus Erythematosus and Rheumatoid Arthritis patients⁴.

Systemic sclerosis is a connective tissue disease characterized by vasculopathy, fibrosis, and progressive autoantibody formation, which can involve multiple organs and skin in various areas of the body. This disease is generally divided into two types, namely the diffuse type and the limited type. Diffuse systemic sclerosis has several markers, including widespread skin fibrosis starting from the fingertips and extremities, and can reach the body. This disease has a fast progression and potentially leads to rapid heart, lung, and kidney damage. Whereas in limited systemic sclerosis, the typical marks of skin fibrosis only appear in the lower extremities, including sclerodactyly, telangiectasia, and calcinosis, and it has a slow progression^5-7.

Skin involvement in systemic sclerosis occurs in > 90% of patients with limited systemic sclerosis and in 100% of patients with diffuse systemic sclerosis. The thickening of the skin is bilateral, which is a hallmark of systemic sclerosis that distinguishes it from other connective tissue disorders. Skin involvement begins at the fingertips and gradually ascends to the proximal extremities⁸. Systemic sclerosis patients with skin fibrosis often experience functional and aesthetic disturbances, which potentially decrease their quality of life^8,9.

The diagnosis of systemic sclerosis is made according to the 2013 European League Against Rheumatism (EULAR) criteria and the American College of Rheumatology (ACR) criteria. These criteria consist of seven clinical criteria that the patient may experience and one laboratory test criterion, namely the presence of specific autoantibodies against systemic sclerosis. The seven clinical criteria include skin thickening on both hand fingers extending to the metacarpophalangeal joints, skin thickening on fingers, lesions on the fingertips, telangiectasia, abnormal nailfold capillaries, pulmonary arterial hypertension (PAH) and/or interstitial lung disease. (ILD), and Raynaud's phenomenon.These criteria have a certain score, and the diagnosis of systemic sclerosis can be made if the total score for the 2013 EULAR criteria and ACR criteria is > 9^10,11.

The modified Rodnan skin score is currently utilized by clinicians to determine the severity of skin involvement in systemic sclerosis patients. This score is calculated based on skin stiffness in 17 body areas, including fingers, hands, forearms, upper arms, thighs, calves, and feet, assessed by palpation and pinching. These seven areas are tested and measured separately on the right and left sides so that they cover 14 areas. The other three areas include face, chest and abdomen. This test is relatively easy to do by experienced clinicians, but the assessment is subjective^10,12.

The excessive formation of fibrous tissue in systemic sclerosis patients is a complex process, involving various immune system cells, including macrophages, T cells, and B cells, as well as involving various cytokines, including transforming growth factor-beta (TGF-β), interleukin-4 (IL-4), interleukin-6 (IL-6), and interleukin-13 (IL-13)¹³.

Transforming growth factor-beta is a central mediator in the formation of fibrous tissue. The method to measure TGF-β levels in serum is the enzyme-linked immunosorbent assay (ELISA), which is more objective than the modified Rodnan’s skin score^14,15.

Studies measuring both TGF-β levels and markers of TGF-β activities in systemic sclerosis patients have shown controversial results, but most have demonstrated that TGF-β levels correlate with the severity of skin involvement.

The roles of TGF-β in the process of vasculopathy have not been clearly discovered, but TGF-β plays a role in endothelial cell and vascular smooth muscle homeostasis, as well as in the formation of fibrous tissue. On that ground, it is assumed that systemic sclerosis therapy might be pursued by inhibiting TGF-β activities¹⁶. The purpose of this study was to determine the correlation between TGF-β levels and the severity of skin involvement in systemic sclerosis patients as determined by the modified Rodnan’s skin scores.

METHODOLOGY:

This study is a correlative observational study with a cross-sectional design. The subjects of this study include sclerosis patients treated at the Rheumatology out patient’sclinic of Hasan Sadikin General Hospital, Bandung. The diagnosis of systemic sclerosis was usethe 2013 EULAR/ACR criteria.This research has received ethical approval number LB.02.01/X.6.5/218/2021 from the Research Ethics Commission of Universitas Padjadjaran, Bandung.

Subjects and Materials:

The inclusion criteria in this study include adult systemic sclerosis patients who control to the Rheumatology outpatient clinic of Hasan Sadikin General Hospital, Bandung, with completely modified Rodnan’s skin score data. Whereas the exclusion criteria include patients with systemic sclerosis and pulmonary tuberculosis infection markers or a history of pulmonary tuberculosis, and patients with systemic sclerosis accompanied by malignancy or a history of malignancy.

The test materials include serum classified as stored biological materials from the research entitled "Development of a Systemic Sclerosis Registry System Based on Electronic Medical Records". The materials were stored at -80ºC.

MRSS and TGF-β Level Tests:

The modified Rodnan’s skin score was assessed and validated by Rheumatologistat Rheumatology Division. The TGF-β level test utilized the micro-ELISA method with stored serum as the materials.

Data Analysis:

The data were analyzed using the Statistical Package for the Social Science (SPSS) version 25. The Saphiro Wilk's Test was adopted for the normality test, while the Spearman Rank Correlation Test was adopted for the correlation test.

RESULTS:

Shapiro Wilk's test was adopted for the normality test on 33 subjects who met the inclusion and exclusion criteria.The results of the normality test on TGF-β levels and age were normally distributed (with a p-value of 0.219 for TGF-β and a p-value of 0.53 for age) so that both were presented in the form of mean + standard deviation (SD). The data on the characteristics of the subjects are presented in Table 1 below.

Table 1. Subject Characteristics Data.

Variables

n (%)

Mean + SD

Age (years)

18-30

31-50

> 50

Sex

Female

Types of systemic sclerosis

Diffuse

Limited

Other organ involvement*

Skin involvement

Raynaud's phenomenon

Lung involvement

Types of therapy*

Cyclophosphamide

Methylprednisolone

Methotrexate

Mycophenolate Mofetil

Azathioprine

Cyclosporine

Diltiazem

Nifedipine

ANA immunofluorescence patterns

Nucleolar homogeneous

Nucleolar

Speckled

ANA Profile*

Anti-scl-70

Anti-RNA Polymerase III

Anti centromere

ANA titer

1:100

1:320

1:1000

1:3200

1:10.000

8 (24.2)

16 (48.5)

9 (27.3)

33 (100.0)

19 (57.6)

14 (42.4)

33 (100.0)

25 (75.6)

20 (60.6)

27 (81.8)

30 (90.9)

26 (78.8)

10 (30.3)

2 (6.1)

1 (3.0)

14 (42.4)

6 (18.2)

21 (63.6)

11 (33.3)

1 (3.1)

29 (87.9)

4 (12.1)

2 (6.1)

1 (3.0)

10 (30.3)

20 (60.6)

40 + 13

Description: SD = Standard Deviation, n = frequency, % = percentage, DMARD = Disease-modifying antirheumatic drug.

*more than one type can apply to one subject

TGF-β level test results:

The serum TGF-β levels of the subjects are presented in Figure 1 below.

Figure 1: Scatterplot of TGF-β Levels with modified Rodnan’s Skin Scores.

Note: x-axis = TGF-β level (ng/mL),

y-axis = modified Rodnan’s skin score

The data generated from this study included TGF-β levels and modified Rodnan’s skin scores. The data are presented in Table 2 below.

Table 2. Distribution of TGF-β levels in each Modified Rodnan’s Skin Score group.

Variables

N (%)

Mean + SD

TGF-β Levels (ng/mL)

Modified Rodnan’s skin scores

1 - 14

15 - 29

30 - 40

> 40

13 (39.4)

19 (57.6)

1 (3.0)

19.6 + 4.7

17.0 + 3.3*

21.4 + 4.8*

18.7*

Description: SD = Standard Deviation, N = Total, % = Percentage *Mean TGF-β levels in each modified Rodnan’s skin score group

Correlation Between Transforming Growth Factor-beta Levels and Modified Rodnan’s Skin Scores:

The statistical analysis results with Spearman's correlation test indicated a positive moderate correlation between TGF-β levels and modified Rodnan’s skin scores (r = 0.513 with p < 0.05). The analysis results denoted that the higher the TGF-β level in the serum, the higher the modified Rodnan’s skin score.

Additional Analysis:

Additional analysis was performed to compare the TGF-β levels of the subjects with diffuse systemic sclerosis and of those with limited systemic sclerosis. Table 3 below illustrates the comparison between the TGF-β levels of the two groups.

Table 3: The comparison between the TGF-β levels of the subject groups with diffuse and limited systemic sclerosis.

Variables

TGF-β Levels

N (%)

Mean (+ SD)

P-value

Types of Systemic Sclerosis

Diffuse

Limited

19 (57.6)

14 (42.4)

20.1 + 4.7

18.5 + 4.6

0.310*

Description: the analysis utilized unpaired T-test, *p<0.05 = significant

Table 3 illustrates that the mean TGF-β level in the diffuse systemic sclerosis group was higher than that in the limited systemic sclerosis group, but the difference was not statistically significant.

DISCUSSION:

All of the 33 subjects who met the inclusion and exclusion criteria were women. These results were in line with a meta-analysis study conducted by Bergamasco et al in 2019 which stated that the female/male ratio in the European population was between 3.8-11.5:1 and in the North American population 4.6-15:1². In a study by Dantas et al. in Brazil in 2016, 94.6% of the subjects were female¹⁷. Meanwhile, in a study conducted by Wakhlu et al. in India in 2017, 96.4% of the subjects were female¹⁸. It can occur due to several factors, including genetic and hormonal factors. The first factor is associated with the X chromosome as it is assumed to play a role in the immune system and the mutation of which is one of the etiological factors of systemic sclerosis. At the same time, women have a pair of X chromosomes while men only have one, thereby the incidence of X chromosome mutations occurs more frequently in women. Such mutations may lead to more severe clinical manifestations¹⁹. Another factor is related to the roles of the estrogen hormone, which is the dominant sex hormone in women. This hormone has been observed to stimulate the production of a number of fibroblast growth factors in vitro and increase the secretion of TGF-β1 by skin tissue fibroblasts. In addition to stimulating the production of collagen in the extracellular matrix, TGF-β also inhibits the degradation of the extracellular matrix^20,21.

Soldano et al. in their study in the United States in 2010 stated that estrogen was able to stimulate fibroblasts to produce fibronectin, type I collagen, and laminin in the extracellular matrix, which could be decreased by the administration of estrogen receptor inhibitors in the extracellular matrix²⁰.

The mean age of the subjects in this study was 40 years with a standard deviation of +13. In addition, 26.5% of the subjects were aged 16-30 years, 47.0% were aged 31-50 years, and 26.5% were aged > 50 years. It similar with the epidemiological data that the majority of systemic sclerosis patients were in their productive age between 31-50 years. According to the results of a meta-analysis study by Hoffman-Vold in Norway in 1999-2009, the mean age of systemic sclerosis patients at first diagnosed was 47 years²². It was supported by a study by Wakhlu et al. in India in 2017, in which the mean age of the subjects was 39 (+10) years¹⁸, as it was also the case in a study by Dantas et al. in Brazil in 2016, in which the mean age of the subjects was 45.1 (+14.3) years¹⁷. In addition, a study by Alfariz et al. in 2015-2016 at the Hasan Sadikin General Hospital, Bandung, also found that the mean age of systemic sclerosis patients was 40 (+10) years²³.

The mean TGF-β level in this study was 19.6(+4.7) ng/mL, with a range of 9.33-32.33ng/mL. A study by Kucharz et al. in Poland in 2014 also revealed that the TGF-β level of the subjects with systemic sclerosis was 17.3+10.8ng/mL, while that in healthy control individuals was 15.6+13.2ng/mL²⁴. Besides, a study conducted by Wakhlu et al. in India in 2018 found that the TGF-β level of the subjects with systemic sclerosis was 0.862+0.443ng/mL, while that in the healthy control individuals was 0.377+0.209 ng/mL¹⁸. The results of the TGF-β level test in this study was similar to that in a study by Kucharz²⁴.

TGF-β is a pleiotropic cytokine that acts on various body cells. This cytokine plays a role in forming fibrous tissue by activating fibroblasts. Activated fibroblasts will differentiate into myofibroblasts, and actived myofibroblasts will form the extracellular matrix. In healthy individuals, if the formation of fibrous tissue is sufficient enough to respond to the wound healing process, the SMAD 3 pathway will inhibit myofibroblast activities. However, in systemic sclerosis patients, such an inhibition process does not occur and thereby fibrous tissue continues to form. In addition, myofibroblast activation in systemic sclerosis is triggered by tissue hypoxia due to vasculopathy, which happens continuously and results in an excessive and uncontrollable formation of fibrous tissue. Accumulation of extracellular matrix will also lead to differentiation of mesenchymal progenitor cells, pericytes, and epithelial cells. This circle process repeats and results in an excessive formation of fibrous tissue, which is one of the hallmarks of systemic sclerosis^25,26.

The mean modified Rodnan’s skin score in this study was 15 and ranged from 2 to 31. A study by Dantas et al. in Brazil in 2016 revealed that the mean modified Rodnan’s skin score of the subjects was 7 and ranged from 0 to 36¹⁷. Meanwhile, Alfaris et al., in their study in Indonesia in 2017, found that the mean modified Rodnan’s skin score of the subjects was 26 and ranged from 2 to 36²³.

This study indicated that TGF-β levels and modified Rodnan’s skin scores had a moderate positive correlation (r = 0.513), which was statistically significant (p = 0.002). The higher the TGF-β level, the higher the modified Rodnan’s skin score in systemic sclerosis patients. Similarly, a study by Kucharz et al. in Poland in 2014 revealed that TGF-β levels correlated with modified Rodnan’s skin scores (r = 0.506)²⁴. It is also supported by a study by Wakhlu et al. in India in 2018, stating that TGF-β levels correlated with modified Rodnan’s skin scores (r=0.35)¹⁸. Although both studies generated similar results, the TGF-β levels ranged differently. It might be caused by the fact that this study utilized reagents from a different factory than those utilized in the study of Wakhlu et al. Besides, the portion of the TGF-β antigen serving as the target of each reagent was not stated in the insert kit. No interference factor was mentioned in the reagents used in this study, but it was stated on the insert kit of reagents used in the study of Wakhlu et al. that they could not eliminate potential interference factors, including the administration of drugs, soluble receptors, and some binding proteins in serum.

TGF-β levels are influenced by several conditions, including the occurrence of other diseases and the administration of several treatments. In this study, patients with malignancy or a history of malignancy and patients with tuberculosis or a history of tuberculosis were excluded. It only included patients with systemic sclerosis who were treated with various types of drugs. The drugs administered for the treatment of systemic sclerosis are mostly immunosuppressants, which may affect the production of TGF-β from inflammatory cells since the main producers of TGF-β are immune system cells¹¹.

Research conducted by Dziadzio et al in the UK in 2005 indicated a different result, stating that TGF-β levels were negatively correlated with the modified Rodnan skin scores.In the study, it was stated that the serum TGF-β level measured was active TGF-β²⁷, while in this study the serum TGF-β level measured was total TGF-β.

Active TGF-β in serum has a short half-life of 2-3 minutes, whereas latent TGF-β has a longer half-life of 100 minutes. Besides, active TGF-β may be concentrated in the target tissues²⁸. These factors might influence the contradicting results of both studies.

TGF-β is a major mediator in the formation of fibrous tissue, but it is not the only mediator. The formation process of fibrous tissue involves various immune system cells, sucs as T cells, B cells, and macrophages, and also various cytokines, sucs as IL-4, IL-13, and IL-6.

This study did not discuss the duration of the disease, the type of treatment, and the varying length of treatment. Healthy individuals were not included as the control sample in this study, thereby the TGF-β levels of normal individuals in the subject population were unidentified.

CONCLUSIONS:

Serum TGF-β levels had a moderately significant positive correlation with modified Rodnan’s skin scores in systemic sclerosis patients. TGF-β as the main mediator of fibrous tissue formation in systemic sclerosis patients but not the only one marker of this disease activity. Other parameters, including IL-6, IL-4, and IL-13, are required.

ACKNOWLEDGEMENTS:

This study was supported by an internal research grant from Universitas Padjadjaran Bandung, Indonesia.

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Received on 04.01.2023 Modified on 13.03.2023

Research J. Pharm. and Tech 2023; 16(12):5754-5759.

DOI: 10.52711/0974-360X.2023.00931