Relationship Between Concentrations Of Interleukine 6 And Tnf-α In Patients With Systemic Lupus Erythematosus Before And After Treatment - Ke Thi Lan Anh

Journal of military pharmaco-medicine n o 5-2019 186 RELATIONSHIP BETWEEN CONCENTRATIONS OF INTERLEUKINE 6 AND TNF-α IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS BEFORE AND AFTER TREATMENT Ke Thi Lan Anh1; Nguyen Van Doan2; Do Minh Trung3 SUMMARY Objectives: To describe levels of serum IL-6 and TNF-α in patients with systemic lupus erythematosus before and after treatment of acute crisis. Subjects: 34 acute systemic lupus erythematosus patients who were treated at the Center for Allergy - Clinical Immunology of Bachmai Hospital from April 2014 to December 2014 and 31 normal controls. Methods: A longitudinal study, levels of IL-6 and TNF-α in serum samples from 34 patients with systemic lupus erythematosus and 31 normal controls were determined by enzyme linked immunosorbent assay. Results: The average age of study participants was 28 ± 10.61. The ratio of females to males was 5.8. Before treatment, the concentration of reduced C3 was reported in 94.11% of patients and the concentration of reduced C4 was present in 79.41% of patients, the C3 concentrations were reduced in 55.88% of patients after treatment and the C4 concentrations were reduced in 20.58% of patients after treatment. The average SLEDAI score before treatment was high: 16.56 ± 3.91, which decreased after treatment: 9.24 ± 5.2 (p < 0.05). 79.41% of patients were responsive to treatment and 20.58% were non-responsive. The group of patients responds to treatment: the average of TNF-α was 47.26 ± 30.32 pg/mL and 18.71 ± 10.71 pg/mL (p < 0.05) before and after treatment, respectively. IL-6 concentration before treatment was 13.78 ± 19.66 pg/mL; after treatment was 0.76 ± 2.21 pg/mL (p < 0.05), there was no difference between IL-6 serum of patients after treatment and control group (1.3 ± 1.83 pg/mL). The group of patients without response to treatment: the average of IL-6 after treatment was 18.43 ± 40.74 pg/mL. TNF-α was 92.71 ± 132.65 pg/mL, they were similar to pre-treatment group (IL-6: 4.31 ± 5.41 pg/mL and TNF-α: 39.42 ± 26.19 pg/mL), p > 0.05. Conclusions: Serum TNF-α and IL-6 are sensitive markers for systemic lupus erythematosus disease activity. They may be useful independent markers for prediction of systemic lupus erythematosus disease activity and to differentiate normal subjects from those having systemic lupus erythematosus. * Keywords: Systemic lupus erythematosus; Cytokine; Interleukin-6; TNF-α. INTRODUCTION Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease with a wide range of clinical presentations which has an effect on multiple organ systems. Patients with lupus experience a loss of self-tolerance as a result of abnormal immunological function and the production of autoantibodies, 1. Haiphong University of Medicine and Pharmacy 2. Hanoi Medical University 3. Vietnam Military Medical University Corresponding author: Ke Thi Lan Anh (kelananh1980@gmail.com) Date received: 25/03/2019 Date accepted: 22/05/2019 Journal of military pharmaco-medicine n o 5-2019 187 which lead to the formation of immune complexes that may adversely affect normal tissue. Although the precise etiologic mechanism is unknown, genetic, hormonal, and environmental factors, as well as immune abnormalities, have been identified. Associations between lupus onset and age, sex, geography, and race have also been established [2, 3]. Cytokines are central factors in both innate and adaptive immunity. They are mostly synthesized by immune cells and in turn participate in the differentiation, maturation, and activation of diverse immune and hematopoietic cells. Abnormalities of various cytokines have been identified in patients with SLE and in murine lupus models. There is growing evidence of the important role of cytokines in SLE pathogenesis, as well as their utility as biomarkers and targets in new therapies. Particularly high levels of interleukine (IL) 6 and TNF-α increased during acute episodes [2, 3]. Therefore, this study was carried out with the following objectives: To describe levels of serum IL-6 and TNF-α in patients with SLE before and after treatment of acute crisis. SUBJECTS AND METHODS 1. Subjects. Our study included 34 acute SLE patients who were treated at the Center for Allergy - Clinical Immunology of Bachmai Hospital from April 2014 to December 2014 and 31 matched healthy control individuals. * Diagnostic criteria for SLE: Patients in the study were diagnosed according to American College of Rheumatology (ACR) in 1997 [3, 4], including 11 criteria: - Malar rash. - Discoid rash. - Photosensitivity. - Oral or nasal ulcer. - Arthritis. - Serositis. - Renal: proteinuria > 0.5 g/24 hour; hematuria, oval fat bodies and fatty casts in the urine. - Neurology. - Hematology: Hemolytic anemia; leukopenia < 4 G/L; thrombocytopenia < 100 G/L. - Immunologic abnormalities: anti-ds- DNA (+); anti-phospholipid (+); anti-Sm (+). - ANA (+). * Initial evaluation based on SLEDAI criteria: The SLEDAI was evaluated as follows: - Inactive disease: SLEDAI = 0 points. - Mildly active disease: SLEDAI ≤ 5 points. - Average active disease: SLEDAI = 6 - 12 points. - Strongly active disease: SLEDAI > 12 points. 2. Methods. * Study design: Longitudinal study. * Study content: - Control group: Taking history, taking previous history, examination: to exclude acute and chronic diseases. Quantification of serum TNF-α, IL-6. Journal of military pharmaco-medicine n o 5-2019 188 - Patient: Each patient was provided with a medical record. + Taking history, taking previous history, examination: Fever, malar rash, fatigue, weight loss, joint pain, hair loss, blood pressure.... + Test: Quantification of serum TNF-α, IL-6 as control group. + After 4 weeks: Physical examination and quantification of serum TNF-α, IL-6. * Tests: It is done at Bachmai Hospital and Vietnam Military Medical University. RESULTS Table 1: Age, gender of the patients. Parameters Female Male Overall ± SD n ± SD n ± SD n Patients 27.03 ± 10.28 29 33.6 ± 11.99 5 28 ± 10.61 34 Control group 35.45 ± 7.54 22 29.89 ± 7.97 9 33.84 ± 7.96 31 Table 2: Change of C3, C4. Biological index Before treament After treament p ± SD n % ± SD n % C3 (mg/dL) Normal (0.9 - 1.8) 0.99 ± 0.056 2 5.89 1.15 ± 0.26 15 44.12 p1:2 < 0.05 Reduction (< 0.9) 0.35 ± 0.13 32 94.11 0.62 ± 0.2 19 55.88 Overall 0.39 ± 0.19 (1) 34 100 0.86 ± 0.36 (2) 34 100 C4 (mg/dL) Normal (0.1-0.4) 0.13 ± 0.036 7 20.59 0.18 ± 0.062 27 79.41 p3:4 < 0.05 Reduction (< 0.1) 0.037 ± 0.02 27 79.41 0.06 ± 0.02 7 20.59 Overall 0.05 ± 0.046 (3) 34 100 0.16 ± 0.08 (4) 34 100 Table 3: Change of SLEDAI. SLEDAI score Before treament After treament p ± SD n % ± SD n % ≤ 5 0 0 0 3.38 ± 1.68 8 23.53 p1:2 < 0.05 6 - 12 10.6 ± 2.6 5 14.7 8.67 ± 1.97 18 52.94 > 12 17.59 ± 3.11 29 85.3 16.38 ± 4.13 8 23.53 Overall 16.56 ± 3.91 (1) 34 100 9.24 ± 5.2 (2) 34 100 Journal of military pharmaco-medicine n o 5-2019 189 79.41 20.58 Responder Non Responder Figure 1: Treatment outcome. Table 4: Serum level of IL-6 and TNF-α responder subgroup. Cytokine Before treament ± SD After treament ± SD Control group ± SD p IL-6 (pg/mL) 13.78 ± 19.66 (1) 0.76 ± 2.21 (2) 1.3 ± 1.83 (3) p(1:2) < 0.05 p(2:3) > 0.05 TNF-α (pg/mL) 47.26 ± 30.32 (4) 18.71 ± 10.71 (5) 5.62 ± 5.8 (6) p(4:5) < 0.05 p(5:6) < 0.05 Table 5: Serum level of IL-6 and TNF-α non-responder subgroup. Cytokine Before treament ± SD After treament ± SD Control group ± SD p IL-6 (pg/mL) 4.31 ± 5.41 (1) 18.43 ± 40.74 (2) 1.3 ± 1.83 (3) p(1:2) > 0.05 p(2:3) > 0.05 TNF-α (pg/mL) 39.42 ± 26.19 (4) 92.71 ± 132.65 (5) 5.62 ± 5.8 (6) p(4:5) > 0.05 p(5:6) > 0.05 Figure 2: ROC analysis of TNF-α and IL-6 in SLE. Journal of military pharmaco-medicine n o 5-2019 190 DISCUSSION In all studies reviewed, females had a higher incidence of SLE compared with males, female/male ratio: 5.8. The mean age: 28 ± 10.61. The UK estimated SLE incidence: 7.89/100,000 person/years (95%CI: 7.46 - 8.31) for females compared with 1.53/100,000 person/year (95%CI: 1.34 - 1.71) for males [5]. Nguyen Huu Truong (2017) conducted a study on 128 patients with SLE at the Center for Allergy - Clinical Immunology of Bachmai Hospital with 92.9% of female patients. The mean age was 31.1 ± 9.46 [1]. C3 was a common factor in both pathways of complement activation, C4 engaged in classical complement activation pathway. Both C3 and C4 were sensitive to lupus diagnosis recommended by ACR as a standard of diagnostic criteria [3]. Before treatment: the concentration of C3 reduced down to 94.11%, that of C4 was decreased in 79.41% of patients, the C3 concentrations were reduced in 55.88% of patients after treatment and the C4 concentrations were reduced in 20.58% of patients after treatment. The mean SLEDAI score before treatment was 16.56 ± 3.91 points and decreased after treatment (9.24 ± 4.0), p < 0.05. Also in the study by Li-jun Song (2013), mean SLEDAI score before treatment was 11.45 ± 3.76 and after treatment, it decreased down to 7.9 ± 4.0 [7]. Our research was similar to Feng Qiu et al’s (2013): SLEDAI scores ranged from 10 to 23 (15.1 ± 3.8) before treatment and from 6 to 22 (12.2 ± 4.0) after treatment [8]. There were 79.41% of patients responding to treatment and 20.58% of patients were non-responsive. The group of patients responded to treatment: The average of TNF-α was 47.26 ± 30.32 pg/mL and 18.71 ± 10.71 pg/mL, respectively (p < 0.05) before and after treatment, p < 0.05. Before treatment, the average of IL-6 was 13.78 ± 19.66 pg/mL and post-treatment it was 0.76 ± 2.21 pg/mL, the difference was statistically significant (p < 0.05), there was no difference between IL-6 serum after treatment and control group (1.3 ± 1.83 pg/mL). The group of patients did not respond to treatment: The average IL-6 after treatment was 18.43 ± 40.74 pg/mL, TNF-α was 92.71 ± 132.65 pg/mL, they were similar to pre-treatment (IL-6: 4.31 ± 5.41 pg/mL and TNF-α: 39.42 ± 26.19 pg/mL) (p > 0.05). In the study by Feng Qiu et al (2013): thirty newly diagnosed severe SLE patients and 30 healthy subjects found that the average concentration of TNF-α patients pre-treatment was 89.59 ± 20.27 ng/mL, after treatment the concentration reduced down to 54.31 ± 17.17 ng/mL, the average concentration of the control group was 37.46 ± 11.63 ng/mL, the difference was statistically significant (p < 0.05). Before treatment, the average concentration of IL-6 serum was 1.56 ± 1.18 pg/mL, after treatment, the average concentration decreased to 0.97 ± 0.37 pg/mL, the average concentration of the control group was 0.7 ± 0.05 pg/mL - the difference was statistically significant (p < 0.05) [8]. Our research was consistent with Li-jun Song’s: before treatment, the mean level of IL-6 was 0.86 (0.7 - 1.72). After treatment, it was 0.72 (0.58 - 1.18) [3]. We tried to analyze biomarkers to see whether IL-6 and TNF-α were predictors of differentiation between healthy people and patients with SLE. ROC analysis of TNF-α and IL-6 was used to assess the discrimination between patients with SLE and controls, the area under the curve (AUC) for TNF-α was 0.98, IL-6 was 0.664 (p < 0.001). In the study by Helena Journal of military pharmaco-medicine n o 5-2019 191 Idborg (2018): the AUC for TNF-α was 0.86 (0.83 - 0.89) [9]. They may be useful independent markers for prediction of SLE disease activity and to differentiate normal subjects from those having SLE. They were increased in the acute phase of the disease. We propose that the TNF- α and IL-6 merit further investigations as clinically useful biomarkers in SLE. CONCLUSIONS From our study, by following up 34 patients with SLE treated in acute crisis, we draw some conclusions: There were 79.41% of patients responding to treatment and 20.58% of patients non-responsive. The group of patients responds to treatment: The average of TNF-α quantity after treatment (18.71 ± 10.71 pg/mL) was reduced in comparison with that of pre-treatment (47.26 ± 30.32 pg/mL). The concentration of IL-6 before treatment was 13.78 ± 19.66 pg/mL which reduced to 0.76 ± 2.21 pg/mL after treatment, the difference was statistically significant (p < 0.05). The group of patients did respond to treatment: The average of IL-6 after treatment was 18.43 ± 40.74 pg/mL, TNF-α was 92.71 ± 132.65 pg/mL, they were similar to pre-treatment group (IL-6: 4.31 ± 5.41 pg/mL and TNF-α: 39.42 ± 26.19 pg/mL), p > 0.05. ROC analysis of TNF-α and IL-6 was used to assess the discrimination between patients with SLE and controls. The AUC for TNF-α was 0.98, IL-6 was 0.664 (p < 0.001). Serum TNF-α and IL-6 are sensitive markers for SLE disease activity. REFERENCES 1. Nguyen Huu Truong. Study the correlation between the disease activity index and some autoantibodies in systemic lupus erythematosus. 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