Correlative Study Of Serum Th1/Th2 Cytokines Levels In Patients With Systemic Lupus Erythematosus With SLEDAI
Manal El- Sayed*, Eman Nofal*, Sahar Al Mokadem*, Inas Al Makhzangy*, Hala Gaballah*** and Hossneia Akl**
Egyptian Dermatology Online Journal 4 (1):
3, June, 2008.Mail to:
|Click Here for a Printable PDF version|
The aim of this study is to investigate the imbalance between Th-1 and Th-2 cytokines in systemic lupus erythematosus patients (SLE) and to assess if any of these cytokines could be related to disease activity.
Twenty three SLE patients and 11 healthy individuals were rolled in this study. Blood samples were collected to evaluate, using ELISA method serum levels of cytokines .Th-1 type: Interferon (IFNg, tumor necrosis factor (TNFa) and Th-2 cytokines: Interleukin (IL-4, IL- 10). Disease activity was assessed using the SLE disease activity index (SLEDAI).
Levels of IFN-g were significantly higher in patients than in healthy controls (P<0.01), as well as IFN/ IL-10, IFN/ IL4, TNF/ IL-10, and TNF/ IL-4 ratios (P<0.01), suggesting a major participation of Th1 over Th2 cytokines. Nevertheless, a direct correlation between Th1 (IFN-g and TNF-a ) and Th2 (IL-4 and IL-10) cytokines was observed in patients (P<0.001), indicating a mutual Th1- Th2 participation. TNF-a levels and the TNF/IL-10 ratio were higher in patients with inactive disease compared with patients with active disease and controls (P<0.05). IL-10 levels were associated with anti-DNA response (P<0.001).
Our results show that Th1 as well Th2 cytokines can be elevated in SLE patients suggesting that lupus is a complex disease that may involve different cytokine patterns in different time points. Also, suggest that high level of TNF-a could be a protective factor in SLE patients, and IL-10 influences the autoimmune response (autoantibody production).
Systemic lupus erythematosus (SLE) is a heterogenous, chronic autoimmune disease characterized by the deposit of immune complexes in different organs. The disease primarily affects women between the third and fourth decades of life. Even though the etiology of SLE is unknown, many predisposing factors have been found, including genetic, environmental, infections, and hormonal factors .
The immune dysregulation that lead to overt SLE is complex but has two main characteristics. One is the peculiar lymphocyte hyperactivity accompanied by immunoglobulin repertoire changes leading to an increased production of autoantibodies . The other is the impaired cell mediated immunity which results from both T lymphocyte and antigen- presenting cell abnormalities [2,3].
The abnormality in the T- cell response is manifested by an imbalance in the production of cytokines. Cytokines have been functionally divided into 2 subgroups: Th1, mainly interleukin (IL)-2, IL-12, interferon (IFN)g , and tumor necrosis factor (TNF) a and b, which mainly activate the cellular machinery of the immune system, and Th2 (IL-4, IL-5, IL-10, and IL-13) cytokines, which activate the humoral machinery [4,5,6,7].
IL-10 is a potent inducer of B lymphocyte differentiation, as well as inhibitor of helper T cell and antigen presenting cell function [8,9]. Some have proposed that the immunologic imbalance in SLE may be related to an abnormally high production of IL-10. lymphocyte hyperactivity may result from autocrine and paracrine effects of IL-10 signaling [10,11].
In patients with SLE, - cell hyperactivity has been associated with a high production of Th2 cytokines, leading to excessive autoantibody production. However, the participation of Th1 cytokines has been equally demonstrated. Both Th1 and Th2 cytokines can participate in promoting or inhibiting autoimmune diseases; thus, a clear-cut distinction between Th1 and Th2 patterns is not without complexity .
In the present study, the relationships between the levels of Th1 (IFNg, TNFa )- Th2 cytokines (IL-10, IL-4) with autoimmune response and disease activity of SLE in a group of patients was evaluated.
Patients And Methods
Twenty three patients with SLE (21 female, 2 male, mean age was (38.2±15.8) who fulfilled 4 or more of the American college of Rheumatology criteria for the classification of SLE  were studied.
No patients fulfilling these criteria were excluded.Patients were on treatment with: prednisolone 10 5mg (80% of patients), hydroxychloroquine 250 mg (45%), azathioprine 50-150 mg (10%), either singularly or in combination.
Eleven healthy people unrelated to the patients, without inflammatory or autoimmune disease as normal control subjects (10 female, 1 male; mean age was 39.1±14.2) were studied.
The SLE Disease Activity Index was developed in Canada and has 24 items. It generated a weighted index of 9 organ systems for disease activity in SLE as follows: 8 for central nervous system and vascular, 4 for renal and musculo-skeletal, 2 for serosal, dermal, immunologic, and 1 for constitutional and haematological affections. The maximum theoretical score is 105, but in practice, few patients have scores greater than 45 . This index produces a global score ranging from 0 to 105 and weighting is used resulting in an individual item score ranging from 1 to 8. A manifestation is recorded if it is present over the past 10 days regardless of severity or whether it has improved or worsened. A previous study has shown that a score of >6 is consistent with active disease requiring treatment . In this study, two stages of SLE were considered as a function of the SLEDAI score; inactive SLE when SLEDAI 6 points and active SLE when SLEDAI >6 points.
Blood samples from all patients and controls were taken during morning. Measurements of IL-4, IL-10, TNF-a and IFN-g in serum samples collected from patients and controls were performed as follows:
Antinuclear antibodies (ANA), anti- DNA were determined by indirect immuno fluorescence.
Data were entered, checked and analyzed by using EPI-INFO (2005). Data were expressed as mean ±standard deviation for quantitative variable, number and percentage for quantitative ones. T-test, ANOVA and chi-squared test and correlation(r) were used when appropriate. P<0.05 was considered significant .
A total of 23 patients with SLE and 11 healthy controls were studied. Patients were on treatment with prednisone 10mg 5 mg/d, (80% of patients), hydroxychloroquine 250mg/d (45%), as azathioprine 50-150 mg/d (10%). With regard to disease activity, 10 patients had inactive disease and 13 had active disease (Table 1).
SLE patients had higher levels of IFN-g , and TNF-a than controls (P<0.01). They also had higher levels of IL-10 but not reach the significant levels than normal controls (P=0.06) (Table 2).
No significant differences were seen with respect to Th2 response. No correlation between disease duration and the cytokines levels or antibody titers was found.
IL-10 levels were significantly higher in patients with anti- DNA. In addition, a direct correlation between the IL-10/ TNF-a ratio and the levels of anti- DNA (r= 0.57, P< 0.01) antibodies was observed (Table 3)
The 2 groups of SLE patients had significantly higher titers of IFN-g than controls (P<0.01); however, there were no differences in IFN-g titers with respect to disease activity.
TNF-a levels were significantly higher in the inactive and active groups when compared with the controls (P<0.001). Patients with inactive disease had statistically higher levels than those in the active group (P< 0.05). Finally, no significant differences were found in Th2 (IL-4, IL-10) cytokines as a function of disease activity (Table 4).
Table (1): General characteristics of the studied groups:
Table (2): Levels of cytokines and autoantibodies in patients with SLE and in healthy individuals:
Table (3): Pearson correlation between cytokines and autoantibodies in patients with SLE:
r = correlation coefficient.
Table (4): TH1/TH2 cytokines levels in patients with SLE in Function of Disease Activity
SLE is a disease characterized by variable autoimmune inflammatory tissue destructions. Defective T cell regulation has been postulated to play a crucial role in its pathogenesis and in the disease manifestations. However, the predominance of Th1 or Th2 cytokines in SLE has not been well defined, and the mechanisms that lead to the aberrant auto-inflammatory syndrome are not clearly understood . In the present study we evaluated the relationship between Th1- Th2 cytokine levels with the production of auto antibodies and the activity of SLE. Our results showed a mutual participation of Th1 and Th2 cytokines in the disease.
TNF-a acutely up regulates the function of immune system, but following prolonged exposure, excessive TNF-a is immunosuppressive. We found that TNF-a levels were diminished as a function of disease activity, suggesting a possible protective role in SLE as has been previously reported [19,20,21]. TNF-a acts as a 2- face cytokine in SLE. First, it could be an immunosuppressive mediator, chronically produced as a defense mechanism or acting as a suppressor of autoantibody synthesis at the T- lymphocyte level. Second, it might be a pro-inflammatory factor acutely released in the local tissues .
IL-10 is a B- cell stimulatory cytokine that also inhibits type 1 cytokine response [23,24]. Several lines of evidence suggest that IL-10 plays a critical role in the immuno-pathogenesis of SLE. Although previous studies have suggested pathogenic roles for raised level of IL-10 in SLE, any involvement of this cytokine with specific clinical manifestation has remained unclear. The absence of a correlation between levels of IL-10 and active clinical subsets in SLE or overall disease activity scores is in agreement with previous studies [25,26]. We observed a significant correlation between IL-10 levels and anti-DNA antibodies-which has been previously reported as supporting a role for IL-10 in active disease in SLE. Serum IL-10 levels have been shown to correlate with overall disease activity when serial measurements were taken in the same patients' overtime .
IFN-g is produced principally by T cells, CD 4+ as well as CD 8+ and natural killer cells. Its main function is the activation of macrophages in innate and acquired response. Its activity increases in the presence of TNF-a and TNF-b . In this study we observed significant increase of IFN-g in patients than controls, although no association to disease activity was found.
Previous studies showed an increase in IFN-g and IL-18 concentration, and there was a positive correlation between both  .IFN- g showed a negative correlation with Th2 cytokines (IFN-g with IL-4 and IL-10), P<0.001.
IL- 4 is produced by a subpopulation of activated T- lymphocytes (Th2). In our study, we did not find significant differences in IL-4 levels between SLE patients and controls, nor was there a statistical difference between the levels of this cytokine and disease activity. However, a direct correlation between IL-4 and the levels of Th1 cytokines was noticed. On contrary to other studies have shown similar results or diminished levels , suggesting that IL-4 does not have such a preponderant role in SLE.
Our results showed that Th1 as well Th2 cytokines can be elevated in SLE patients suggesting that lupus is a complex disease. That may be supported by different cytokine patterns in different time- points. In accord with the most recent literature we observed that a sharp distinction between Th1 and Th2 cytokine pattern is not feasible nor meaningful in SLE patients; either because the mechanism regulating this disease is too complex or because too many factors such as disease activity, treatment , organ involvement have to be taken into consideration  .Thus, to extend the findings of the present work, it would be necessary to design longitudinal studies to establish a cause- effect relationship, and to develop methods that not only evaluate cytokine levels, but also determine the function and regulation of their expression, including their gene polymorphism . The results obtained should enhance our understanding of the immunology of SLE and other autoimmune diseases.
1. Alindon TMc, Update on the epidemiology of systemic lupus erythematosus: New spins on old ideas, Curr opin Rheumatol. 12: 104-112, 2000.
2. Block SR, Gibbs CB, Stevens MB and Shulman LE, Delayed hypersensitivity in systemic lupus erythematosus, Ann Rheum Dis. 27: 311-318, 1968.
3. Via GS, Tsokos GC, Bermas B, Clerici M and Shearer GM, T cell-antigen- presenting cell interactions in human systemic lupus erythematosus: evidence for heterogeneous expression of multiple defects, J Immunol. 151: 3914-3922, 1993.
4. Funauchi M, Ikoma S, Enomoto H and Horiuchi A, Decreased Th1-like and increased Th2-like cells in systemic lupus erythematosus, Scand J Rheumatol. 27: 219-224, 1998.
5. Mosmann TR and Coffman RL, TH1 and TH2 cells: Different patterns of lymphokine secretion lead to different functional properties, Annu Rev Immunol. 7: 145-173, 1989.
6. Romagnani S, The Th1/Th2 paradigm, Immunol Today. 18: 263-266, 1997.
7. Viallard JF, Pellegrin JL, Ranchin V, Schaeverbeke T, Dehais J, Longy Boursier M et al., Th1 (IL-2, interferon- gamma (IFN-gamma)] and Th2 (IL-10,IL-4) cytokine production by peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus (SLE), Clin Exp Immunol. 115; 189-195, 1999.
8. Rousset F, Garcia E, Defrance T, Peronne C, Hsu DH, Kastelin R et al., Interleukin-10 is a potent growth and differentiation factor for activated human B lymphocytes, Proc Natl Acad Sci USA. 89; 1890- 1893, 1992.
9. De Waal Malefyt R, Yssel H and de Vries JE, Direct effect of IL-10 on subsets of human CD4+ T cell clones and resting T cells: specific inhibition of IL-2 produciton and proliferation, J Immunol. 150: 4754-4765, 1993.
10. Amel Kashipaz MR, Huggins ML, Lanyon P, Robins A, Powell RJ, Todd I, Assessment of Be1 and Be2 cells in systemic lupus erythematosus indicates elevated interleukin- 10 producing CD5+B cells, Lupus. 12: 356-63, 2003.
11. Amy M. Beebe and Rene de Waal Malefyt, The role of interleukin-10 in autoimmune disease: Systemic lupus erythematosus (SLE) and multiple sclerosis (MS), Science Direct-cytokine & Growth factor Reviews September 2002.
12. Amerio P, Frezzolini A, Abeni D, Teolfoli P, Girardelli CR, De Pita O et al., Increased IL-18 in patients with systemic lupus erythematosus: Relations with Th-1, Th-2, proinflammatory cytokines and disease activity. IL -18 is a marker of disease activity but does not correlate with proinflammatory cytokines, Clin Exp Rheumatol. 20: 535-538, 2002.
13. Tan EM, Cohen AS, Fries JF, Masi AT, MC Shane DJ, Roth field NF et al., The 1982 revised criteria for the classification of systemic lupus erythematosus, Arthiritis Rheum. 25: 1271-1277, 1982.
14. Sdaile E., Abrahamowic ZM., and Joseph L, Laboratory tests as predictors of disease exacerbations in systemic lupus erythematosus, Arth Rheum. 39: 370-378, 1996.
15. Bombardier C, Gladman DD, Vrowitz M, Derivation of the SLE DAI. A disease activity index for Lupus patients, Arth Rheum. 35: 630, 1992.
16. Abrahamowicz M, Fortin PR, Du BR, Nayak V, Neville C, Liang MH, The relationship between disease activity and expert physician's decision to start major treatment in active systemic lupus erythematosus: a decision aid for development of entry criteria for clinical trials, J Rheumatol. 25: 277-84, 1998.
17. EPI-INFO 2005: Soft ware computer package for statistics and epidemiology on microcomputer CDC, Atlanta, USA.
18. Lit LCW, Wong CK, Tam LS, et al., Raised plasma concentration and ex vivo production of inflammatory chemokines in patients with systemic lupus erythematosus, Ann Rheum Disease. 65: 209-215, 2006.
19. Jacob CO, Tumor necrosis factor a in autoimmunity: Pretty girl or old witch?, Immunol Today. 13: 122-125, 1992.
20. Mageed RA and Isenberg DA, Tumour necrosis factor alpha in systemic lupus erythematosus and anti-DNA autoantibody production, Lupus. 11: 850-855, 2002.
21. Gomez D, Correa PA, Gomez LM, et al., Th1/Th2 cytokines in patients with systemic lupus erythematosus: is tumor necrosis factor a protective?, Seminars in Arthiritis and Rheumatism. 33: 404-413, 2004.
22. Aringer M and Smolen JS, Complex cytokine effects in a complex autoimmune disease: Tumor necrosis factor in systemic lupus erythematosus, Arthritis Res Ther. 5: 172-177, 2003.
23. Fitch FW, Lancki DW, Gajewski TF, T-cell mediated immune regulation. In: Paul WE, ed. Fundamental immunology. New York: Raven, 1993: 801-35.
24. Durum SK, Oppenheim JJ, Proinflammatory cytokines and immunity. In: Paul WE, ed. Fundamental immunology. New York: Raven, 1993: 801-35.
25. Lucas JA, Ahmed SA, Casey ML, MacDonald PC, Prevention of autoantibody formation and prolonged survival in New Zealand black/ New Zealand white F1 mice fed dehydroisoandrosterone, J Clin Invest. 75: 2091-93, 1985.
26. Chang DM, Chu SJ, Chen HC, Kuo SY, Lai JH, Dehdroepiandrosterone suppresses interleukin 10 synthesis in women with systemic lupus erythematosus, Ann Rheum Disease. 63: 1623-1626, 2004.
27. Daynes RA, Dudley DJ, Araneo BA, Regulation of murine lymphokine production in vivo. IL. Dehydroepiandrosterone is a natural enhancer of interleukin 2 synthesis by helper T cells, Eur J Immunol. 20: 793-802, 1990.
28. Amerio P, Frezzolini A, Abeni D, Teo Foli P, et al, Increased IL-18 in patients with systemic lupus erythematosus: Relations with Th-1, Th-2, pro-inflammatory cytokines and disease activity. IL-18 is a marker of disease activity but does not correlate with pro-inflammatory cytokines, Clin Exp Rheumatol. 20: 535-538, 2002.
29. Wong CK, Ho CY, Elevation of proinflammatory cytokine (IL-18, IL-17, IL-12) and Th2 cytokine ( IL-4) concentrations in patients with systemic lupus erythematosus, Lupus. 9: 589-593, 2000.
30. Nagy G, Pallinger E, Antal-Szalmas P, et al., Measurement of intracellular interferon- gamma and interleukin-4 in whole blood T lymphocytes from patients with systemic lupus erythematosus, Immunol Letter. 74: 207-210, 2000.
© 2008 Egyptian Dermatology Online Journal