Th2 cytokines in patients with systemic lupus erythematosus: is tumor necrosis factor α protective?

Th1/Th2 Cytokines in Patients with Systemic Lupus Erythematosus: Is Tumor Necrosis Factor ␣ Protective? Diana Go´mez, Paula A. Correa, Luis Miguel Go´mez, Jose´ Cadena, Jose´ F. Molina, and Juan-Manuel Anaya Objectives: To determine the circulating levels of Th1 and Th2 cytokines in patients with systemic lupus erythematosus (SLE) and to elucidate their association with disease activity and autoimmune response. Methods: We included 52 patients and 25 healthy controls. Serum levels of tumor necrosis factor (TNF) ␣, interferon (IFN) ␥, interleukin (IL)-12p70, IL-10, and IL-4, as well as anti-DNA, -Ro, -La, -RNP, and -Sm antibodies were determined by enzyme-linked immunosorbent assay. Disease activity was recorded according to the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) and classified as very active (SLEDAI > 13), moderately active (SLEDAI: 3-12), or inactive (SLEDAI < 2). Results: The mean age of the patients was 34.2 ⴞ 12.6 years, and the mean duration of disease was 4.9 ⴞ 7.6 years. Twelve patients (23%), 20 patients (34.5%), and 20 patients (34.5%) had highly, moderately, and inactive SLE, respectively. Levels of IFN-␥, TNF-␣, and IL-12 were significantly higher in patients than in healthy controls (P < .03), as well as the IL-12/IL-10, IL-12/IL-4, IFN/IL-10, IFN/IL-4, TNF/IL-10, and TNF/IL-4 ratios (P < .01), suggesting a major participation of Th1 over Th2 cytokines. Nevertheless, a direct correlation between Th1 (IFN-␥ and TNF-␣) and Th2 (IL-4 and IL-10) cytokines was observed in patients (r > .5, P < .01), indicating a mutual Th1-Th2 participation. TNF-␣ levels and the TNF/IL-10 ratio were higher in patients with inactive disease compared with patients with very active disease and controls (P < .04). IL-12 levels and IL-12/IL-4, as well as IL-12/IL-10, ratios were higher in patients with very active disease than in those with inactive SLE and controls (P < .01). IL-10 levels were associated with anti-DNA, anti-Ro, and anti-La response (P < .01). Conclusion: Our results suggest that TNF-␣ could be a protective factor in SLE patients, whereas IL-12p70 participates in disease activity and IL-10 influences the autoimmune response (autoantibody production). Semin Arthritis Rheum 33:404-413. © 2004 Elsevier Inc. All rights reserved. INDEX WORDS: Systemic lupus erythematosus; cytokines; TNF-␣; IL-10; IL12; autoantibodies; SLEDAI. From the Cellular Biology and Immunogenetics Unit, Corporacio´n para Investigaciones Biolo´gicas, Medellin, Colombia; and Rheumatology Unit, Clinica Universitaria Bolivariana, School of Medicine, Universidad Pontificia Bolivariana, Medellin, Colombia. Diana Go´mez, BSc: Assistant Researcher, Cellular Biology and Immunogenetics Unit, Corporacio´n para Investigaciones Biolo´gicas, Medellin, Colombia; Paula A. Correa, MSc: Assistant Researcher, Cellular Biology and Immunogenetics Unit, Corporacio´n para Investigaciones Biolo´gicas, and Assistant Professor, School of Medicine, Universidad Pontificia Bolivariana, Medellin, Colombia; Luis Miguel Go´mez, MV: Assistant Researcher, Cellular Biology and Immunogenetics Unit, Corporacio´n para Investigaciones Biolo´gicas, Medellin, Colombia; Jose´ Cadena, MD: Assistant Researcher, Cellular Biology and Immunogenetics Unit, Corporacio´n para Investigaciones Biolo´gicas, and Rheumatology Unit, Rheumatology Unit, Clı´nica 404

Universitaria Bolivariana, Medellin, Colombia; Jose´ F. Molina, MD: Associate Professor, School of Medicine, Universidad Pontificia Bolivariana, Medellin, Colombia Juan-Manuel Anaya, MD: Associate Researcher, Cellular Biology and Immunogenetics Unit, Corporacio´n para Investigaciones Biolo´gicas, and Professor of Medicine, School of Medicine, Universidad Pontificia Bolivariana, Medellin, Colombia. Supported by the Asociacio´n Colombiana de Reumatologı´a, Bogota´, and the Corporacio´n para Investigaciones Biolo´gicas, Medellı´n, Colombia. Address reprint requests to: Juan-Manuel Anaya, MD, Corporacio´n para Investigaciones Biolo´gicas, Cra. 72-A No 78-B141, Medellı´n, Colombia. E-mail: [email protected] © 2003 Elsevier Inc. All rights reserved. 0049-0172/04/3306-0000$30.00/0 doi:10.1016/j.semarthrit.2003.11.002

Seminars in Arthritis and Rheumatism, Vol 33, No 6 (June), 2004: pp 404-413

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S

YSTEMIC LUPUS erythematosus (SLE) is a heterogeneous, 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 playing an important role have been found, including genetic, environmental, infectious, and hormonal factors (1). In SLE, B-cell hyperactivity and the presence of multiple autoantibodies are observed (2,3). Anti-IgG DNA antibodies are present in nearly 70% of patients, and some studies have suggested that they are nephrotoxic (3,4). Another characteristic of patients with SLE is the abnormality in the T-cell response, 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) ␥, and tumor necrosis factor (TNF) ␣ and ␤, 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 (5– 8). In patients with SLE, B-cell hyperactivity has been associated with a high production of Th2 cytokines. However, the participation of Th1 cytokines has been equally demonstrated (9). 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 (10). In SLE, the different results concerning cytokines should be considered depending on the studied model, because discrepancies exist between murine and human (patients) studies (10 –22). In the present study, the simultaneous relationships between the levels of Th1-Th2 cytokines with the production of autoantibodies and the activity of SLE in a group of patients was evaluated. METHODS

Study Population This was a cross-sectional study. SLE patients were enrolled in the Rheumatology Unit at the Clinica Universitaria Bolivariana, Medellin, Colombia. All patients fulfilled 4 or more of the American College of Rheumatology criteria for the classification of SLE (23). Healthy people unrelated to the patients, without inflammatory or autoimmune disease, matched to patients by age (⫾5 years), gender, and geography were included as controls. This study was approved by the local ethics committee.

Clinical Variables Information on patient demographics and cumulative clinical and laboratory manifestations over the disease course were obtained either by verification during discussion with the patient or by chart review, and were recorded in a standard data-collection form created for that purpose. Each clinical and laboratory variable was registered as “present” or “absent” for every specific patient during the course of the disease and at the time of blood sample collection. The clinical and laboratory variables associated with SLE, including each feature of the revised American College of Rheumatology criteria (23), were evaluated and defined as follows: 1) arthritis: nonerosive arthritis involving 2 or more peripheral joints, characterized by tenderness, swelling, or effusion; 2) malar rash; 3) photosensitivity; 4) alopecia; 5) discoid lupus; 6) Raynaud’s phenomenon; 7) renal involvement, as evidenced by a renal biopsy result showing the World Health Organization’s (WHO) class II-V histopathology, active urinary sediment, or proteinuria ⬎500 mg/24 h. Nephrotic syndrome was defined as ⬎3.5 g/d of proteinuria, hypoalbuminemia (⬍2.8 g/dL), hyperlipidemia, and edema. Lupus nephritis was defined as present or absent according to the abnormalities of the previous tests. 8) Neurologic involvement, as evidenced by seizures or psychosis without any other definable cause, or other conditions such as peripheral neuropathy, stroke, transverse myelitis, chorea, or other central nervous system lesions directly attributable to SLE in the absence of other causes; 9) pleuritis: pleural rub and/or effusion and/or typical pleuritic pain; 10) pericarditis: documented by electrocardiogram, rub, or evidence of pericardial effusion; 11) autoimmune hemolytic anemia, with a hematocrit ⬍35%, reticulocyte count ⬎4%, and positive Coombs test; 12) leukopenia, white cells ⬍4000/mm3; 13) thrombocytopenia, platelets ⬍100,000/mm3; 14) arterial or venous thrombosis diagnosed on clinical grounds and confirmed by appropriate tests. Comorbility was also recorded, and included the presence or absence of infection, arterial hypertension (blood pressure levels ⬎ 140/90), diabetes mellitus (fasting glycemia ⱖ 126 mg/dl in 2 occasions), coronary disease (history of myocardial infarction, stable or unstable angina), hyperlipidemia (LDL cholesterol ⬎ 130 mg/dl and triglycerides ⬎ 150 mg/dl), and hypothyroidism (TSH ⬎ 5 mU/L).

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Table 1: General Characteristics of 52 Patients with SLE Characteristic Sex, female:male 51:1 Age (y) 34.2 ⫾ 12.6 Duration of SLE (y) 4.9 ⫾ 7.6 Activity of the disease (SLEDAI), % ⱕ2 38.5 3–12 38.5 ⱖ13 23 Clinical Features, % Musculoskeletal 96 Cutaneous 92 Raynaud’s phenomenon 54 Cardiopulmonary 31 Renal 40 Hematological 62 Neuropsychiatric 44 Antibodies, % Antinuclear 94 Anti-DNA 52 Anti-Ro 39 Anti-La 15 Anti-Sm 31 Anti-RNP 62

Disease Activity Disease activity was evaluated according to the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) (24). Three stages of SLE were considered as a function of the SLEDAI score: inactive SLE when SLEDAI ⱕ 2, moderately active SLE when SLEDAI was between 3 and 12, and very active disease when SLEDAI ⱖ 13 (25). Cytokines Measurements of IL-4, IL-10, IL-12(p70), TNF-␣, and IFN-␥ in serum samples collected from patients and controls were performed by solid-phase sandwich enzyme-linked immunosorbent assay using the OptEIA kits (Pharmingen, BD Biosciences, San Diego, CA), according to the manufacturer’s instructions. Autoantibodies Antinuclear antibodies were determined by indirect immunofluorescence using HEp-2 cells as substrate. Serum levels of IgG anti-DNA, anti-Ro,

anti-La, anti-RNP, and anti-Sm antibodies were studied by enzyme-linked immunosorbent assay (QUANTA-Lite, Inova, San Diego, CA), according to the manufacturer’s instructions. Autoantibodies and cytokines were simultaneously determined in the same serum samples. Statistical Analysis The results are presented as means ⫾ standard deviation (SD) and in percentages. The differences in averages were examined by the 2-tailed MannWhitney test. The correlations between levels of cytokines, autoantibodies, SLEDAI, and disease duration were established by the Pearson test. In all cases, P ⬍ .05 was considered significant. Results were analyzed with SPSS software (26) and Graphpad Prism (San Diego, CA) (27). RESULTS

Clinical Characteristics of Patients A total of 52 patients with SLE (Table 1) and 25 controls were studied. Ninety-eight percent of patients were treated with steroids (prednisolone, 12 ⫾ 3 mg/d), 30% were under monthly cyclophosphamide pulses (0.7 g/m2), 20% were taking azathioprine (50-150 mg/d), and 60% were on chloroquine (250 mg/d). With regard to disease activity, the mean Table 2: Levels of Autoantibodies and Cytokines in Patients with SLE and in Healthy Individuals (controls)

Autoantibodies (UI) Anti-DNA Anti-Ro Anti-La Anti-Sm Anti-RNP Cytokines (pg/mL) IL-4 IL-10 IL-12 TNF-␣ IFN-␥ NA, not applicable. *P ⬍ .0001.

SLE (n ⴝ 52)

Controls (n ⴝ 25)

674.5 ⫾ 670.8 33.2 ⫾ 38.9 25.4 ⫾ 47.7 27.9 ⫾ 37.1 50. ⫾ 48.2

NA NA NA NA NA

7.8 ⫾ 13.1 20.4 ⫾ 17.4 380.1 ⫾ 596.8* 86.6 ⫾ 423.7* 31 ⫾ 13.5*

6.44 ⫾ 8.3 27.4 ⫾ 24 31.44 ⫾ 27.4 18.72 ⫾ 14.3 5.3 ⫾ 8

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Table 3: Pearson Correlation Coefficients of Th1–Th2 Cytokines and Autoantibodies in Patients with SLE

IL-4 IL-10 IL-12 TNF-␣ IFN-␥ TNF-␣/IL-10 IL-10/TNF-␣ TNF-␣/IL-4 IL-4/TNF-␣ IFN-␥/IL-10 IL-4/IFN-␥ IL-10/IFN-␥ IFN-␥/IL-4 IL-12/IL-10 IL-10/IL-12 IL-12/IL-4 IL-4/IL-12 Anti-ADN Anti-Ro Anti-La Anti-Sm Anti-RNP

Duration of SLE

IL-4

IL-10

IL-12

TNF-␣

IFN-␥

⫺0,01 ⫺0,03 ⫺0,02 ⫺0,03 ⫺0,13 0,05 ⫺0,10 0,14 0,05 0,00 0,06 0,02 ⫺0,03 0,01 0,01 0,00 0,02 0,04 ⫺0,22 ⫺0,14 ⫺0,19 ⫺0,12

0,81† 0,36† 0,88† 0,62† 0,76† ⫺0,21 0,41† 0,06 ⫺0,31 0,88† 0,41† ⫺0,27 ⫺0,11 0,01 ⫺0,18 0,35* ⫺0,15 ⫺0,11 ⫺0,06 ⫺0,14 0,05

0,28* 0,71† 0,63† 0,57† ⫺0,06 0,34* ⫺0,03 ⫺0,59† 0,68† 0,79† ⫺0,09 ⫺0,20 0,26 0,02 0,33* 0,09 0,01 0,03 ⫺0,07 0,15

0,47† 0,21 0,36† 0,11 0,22 0,15 ⫺0,12 0,22 0,09 0,02 0,79† ⫺0,36† 0,24 ⫺0,29 ⫺0,20 ⫺0,19 ⫺0,07 ⫺0,21 ⫺0,10

0,59† 0,87† ⫺0,12 0,56† ⫺0,15 ⫺0,17 0,58† 0,26 ⫺0,10 ⫺0,03 ⫺0,08 ⫺0,06 0,04 ⫺0,14 ⫺0,10 ⫺0,07 ⫺0,11 0,13

0,53† ⫺0,22 0,41† ⫺0,15 ⫺0,12 0,41† 0,15 ⫺0,08 ⫺0,12 0,11 ⫺0,12 0,19 ⫺0,19 ⫺0,08 ⫺0,11 ⫺0,16 ⫺0,11

*P ⬍ .05. †P ⬍ .01.

SLEDAI score was 7.53 ⫾ 8.2. Twenty patients had inactive disease, 20 had moderately active disease, and 12 had very active disease. Cytokines Levels SLE patients had higher levels of IL-12 (p70), IFN-␥, and TNF-␣ than controls (Table 2) (P ⬍ .001). No significant differences were observed with respect to the Th2 response (Table2). Nonetheless, a direct correlation between Th1 and Th2 cytokines was noticed, indicating a mutual participation of both Th1 and Th2 groups (Table 3, Fig. 1). There was no correlation between disease duration and the cytokines levels or antibody titers (Table 3). IL-10 levels were significantly higher in patients with anti-Ro antibodies (33.6 vs. 22.1 pg/mL, P ⫽ .008). In addition, a direct correlation between the IL-10/TNF-␣ ratio and the levels of anti-La (r ⫽ .44, P ⬍ .01) and anti-DNA (r ⫽ .35, P ⬍ .05) antibodies was observed.

IL-12(p70) levels were significantly higher in patients with Raynaud’s phenomenon than in those without it (29 vs. 18.3 pg/mL, P ⫽ .009). Patients with a history of infection had higher TNF-␣ levels (30.8 vs. 22.5 pg/mL, P ⫽ .05). No association or correlations between cytokines and other clinical or therapeutic parameters were observed. Cytokine Levels and Disease Activity Cytokine levels as a function of disease activity are shown in Table 4. A significant elevation of IL-12 (p70) was observed in all 3 groups compared with controls (P ⬍ .01). Patients with very active disease had significantly higher levels of IL-12 (p70) than those with inactive and active disease (P ⬍ .05). All 3 groups of SLE patients had significantly higher titers of IFN-␥ than controls (P ⬍ .01); however, there were no differences in IFN-␥ titers with respect to disease activity.

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Fig 1.

Mutual participation of representative cytokines of the Th1 and Th2 response in patients with SLE.

Significantly higher levels of TNF-␣ were observed in the inactive and active groups when compared with the controls (P ⬍ .01). Patients with inactive disease had statistically higher levels

than those in the very active group (P ⬍ .01). Finally, no significant differences were found in Th2 (IL-4, IL-10) cytokines as a function of disease activity (Table 4).

Table 4: Th1/Th2 Cytokines and Autoantibody Levels in Patients with SLE in Function of Disease Activity Activity of SLE Characteristics

SLEDAI <2 (n ⴝ 20)

SLEDAI 3–12 (n ⴝ 20)

SLEDAI >13 (n ⴝ 12)

Control Group (n ⴝ 25)

Age (y) Duration of SLE (y) IL-4 (pg/mL) IL-10 (pg/mL) IL-12 (pg/mL) TNF-␣ (pg/mL) IFN-␥ (pg/mL) TNF-␣/IL-4 ratio TNF-␣/IL-10 ratio IFN-␥/IL-10 ratio IL-12/IL-4 ratio IL-12/IL-10 ratio Anti-DNA (UI/mL) Anti-Ro (Units) Anti-La (Units) Anti-Sm (Units) Anti-RNP (Units)

36.3 ⫾ 16.4 5.3 ⫾ 9 11.8 ⫾ 25 22.9 ⫾ 25 303 ⫾ 546† 196 ⫾ 675.5† 32.5 ⫾ 14.8† 6.8 ⫾ 13.6 2.8 ⫾ 7㛳 2.5 ⫾ 1.6† 149 ⫾ 521† 14.5 ⫾ 13.7† 578 ⫾ 589 34 ⫾ 35 12.3 ⫾ 15.1 27.4 ⫾ 38.5 48 ⫾ 52

32 ⫾ 8.1 3.2 ⫾ 4 5.3 ⫾ 7.4 17.6 ⫾ 9.6 324 ⫾ 579† 25.1 ⫾ 69.8† 32 ⫾ 15.4† 3.3 ⫾ 7.8 1.3 ⫾ 4† 2.3 ⫾ 1.6† 61.4 ⫾ 86.4† 30.6 ⫾ 65.3† 581 ⫾ 672.6 36.3 ⫾ 48.4 38.4 ⫾ 63.2 35.1 ⫾ 45 56.3 ⫾ 53

34.8 ⫾ 12.3 7.2 ⫾ 7.6 4.1 ⫾ 4.8 19.6 ⫾ 10 629 ⫾ 726*‡† 8.1 ⫾ 17.4 26.45 ⫾ 5.2† 4.6 ⫾ 10 1 ⫾ 3㛳 1.7 ⫾ 0.8† 574.5 ⫾ 1126† 43.2 ⫾ 51.1† 996.3 ⫾ 787.7 26 ⫾ 30.4 10.8 ⫾ 4.3 17 ⫾ 11 43 ⫾ 34.4

33 ⫾ 9.2 NA 6.4 ⫾ 8.3 27.4 ⫾ 24 31.4 ⫾ 24 19 ⫾ 14.3§ 5.3 ⫾ 8 6.5 ⫾ 8.2 0.9 ⫾ 0.6 0.13 ⫾ 0.1 6.8 ⫾ 6.8 1.4 ⫾ 1.6 NA NA NA NA NA

NA, not applicable. *P ⬍ .05 compared with the SLEDAI ⱕ2 group. †P ⬍ .01 compared with the control group. ‡P ⬍ .05 compared with the SLEDAI 3–12 group. §P ⬍ .05 compared with SLEDAI ⱖ13. 㛳P ⬍ .05 compared with the control group.

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DISCUSSION

We evaluated the relationship between Th1Th2 cytokine levels with the production of autoantibodies and the activity of SLE. Our results show a mutual participation of Th1 and Th2 cytokines in the disease, and suggest that TNF-␣ may play a protective role. The importance of serum IL-12p70 in the activity of disease and of IL-10 in the synthesis of autoantibodies is documented. TNF-␣ is a 17-kd protein containing 157 amino acids (28,29). The major sources of TNF-␣ are the cells of the monocyte/macrophage lineage. T lymphocytes, neutrophils, mast cells, and endothelium also contribute to its synthesis under different circumstances (29). The induction by TNF-␣ of multiple chemokines and adhesion molecules is of major importance in rapidly attracting immune and inflammatory leukocytes to the site of injury and TNF-␣ release. TNF-␣ acutely up regulates the function of the immune system, but following prolonged exposure, excessive TNF-␣ is immunosuppressive (28,29). High levels of this cytokine generate systemic effects that include the induction of fever, the synthesis of acute-phase proteins, as well as cachexia. It also causes intravascular thrombosis and shock (28,29). We found that TNF-␣ levels were diminished as a function of disease activity, suggesting a possible protective role in SLE (Table 4), as has been previously reported (17,30,31). Nearly 10% of patients with rheumatoid arthritis treated with TNF-␣ blockers develop anti-DNA antibodies (31,32), and several patients treated with such blockers developed SLE (33). On the other hand, in populations with a high exposure to infectious diseases, and therefore with a continuous synthesis of TNF-␣, the prevalence of SLE is low (31,34). In our patients, higher levels of TNF-␣ were observed in those with a history of infection (although not statistically significant). Nevertheless, it is possible that TNF-␣ acts as a 2-face cytokine in SLE. First, it could be an immunosuppressive mediator, chronically produced as a defense mechanism (28,29) or acting as a suppressor of autoantibody synthesis at the T-lymphocyte level (35). Second, it might be a proinflammatory factor acutely released in the local tissues (35).

IL-12 is a proinflammatory cytokine that induces the production of IFN-␥, favors the differentiation of Th1 cells, and links innate resistance and adaptive immunity (36,37). Dendritic cells and phagocytes produce IL-12 in response to pathogens during infection. Production of IL-12 is dependent on different regulation mechanisms of expression of the genes encoding IL-12, patterns of Toll-like receptor expression, and crossregulation between the different dendritic cells subsets, involving cytokines such as IL-10 and type I IFN (36,37). Biologically active IL-12 is a 70-kd heterodimeric cytokine (IL-12p70) made from disulfide-linked p35 (a) and p40 (b) chains. p35 is constitutively produced by almost all cell types, whereas p40 expression (and thereby IL12p70 production) is restricted to antigen-presenting cells, neutrophils, keratinocytes and Epstein-Barr Virus–transformed B cells. In our study, we observed that an increase in the production of IL-12p70 was associated with SLE (Tables 2 and 4). Previous studies, similar to our own, have shown diverse results as a function of the studied subunit (Table 5). Our results differ from those of Liu et al (19) and Lauwerys et al (20), who observed the absence of IL-12p70 in patients with SLE. Our study of this cytokine was replicated and the results confirmed in patients as well as in controls. Differences in the capturing antibody and the patients studied might explain these discrepancies. IL-10 is a multifunctional cytokine with diverse effects on most hemopoietic cell types. The principal routine function of IL-10 appears to be to limit, and ultimately terminate, inflammatory responses. In addition to these activities, IL-10 regulates growth and/or differentiation of B cells, natural killer cells, cytotoxic and helper T cells, mast cells, granulocytes, dendritic cells, keratinocytes, and endothelial cells (38). We observed that IL-10 was associated with the synthesis of autoantibodies, as previously reported (Table 6). Other studies have demonstrated a relationship between IL-10 levels and activity of the disease (22). Verthelyi et al (39) did not find a significant difference in IL-10 –secreting cells in SLE patients compared with healthy controls. IFN-␥ is a dimeric glycoprotein with subunits of 146 amino acids. The 2 forms of the active protein are 20 and 25 kd, respectively (28,40). It is produced principally by T cells, CD4⫹ as well as

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Table 5: IL-12 in Patients with SLE

Type

N

Type of Study/Sample

IL-12 (total)

49 20 24

In vitro (PBMC) In vitro (PBMC) In vitro (PBMC)

39 20 36 60 34 40 20 20 28

Serum Serum Serum Serum Serum Serum In vitro (PBMC) In vitro (PBMC) Serum

p40

Results

Reference

Decreased production Increased production Negative correlation between its production and that of IL-10, titers of anti-DNA and disease activity Increased levels Increased levels Increased levels Increased levels Decreased as a function of activity Decreased levels in nephritis Decreased production Gene expression diminished Increased levels and correlation with disease activity

11 15 12 13 10 14 16 17 18 19 19 20

PBMC, peripheral blood mononuclear cells.

CD8⫹, 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-␣ and TNF-␤ (28). We observed an increase of IFN-␥ in patients with respect to controls, although no association to disease activity was found. Previous studies showed an increase in IFN-␥ in SLE, and an association with the synthesis of IL-18. Nevertheless, results differ with regard to the association of its levels and disease activity (Table 7). IL-4 is a glycoprotein of 129 amino acids (20

kd) produced by a subpopulation of activated T lymphocytes (Th2). It contains 6 residues of cystein involved in the formation of disulfide bridges. These bonds are essential to the biological activity of the molecule. IL-4 promotes the proliferation and differentiation of activated B cells and augments the expression of class II molecules of the major histocompatibility complex. The latter helps B lymphocytes respond to stimuli proceeding from other B cells and aids in presenting antigens to T cells. This cytokine inhibits the activation of natural killer cells and stimulates the proliferation of

Table 6: IL-10 in Patients with SLE

N

Type of Study/ Sample

49 24 20

In vitro (PBMC) In vitro (PBMC) In vitro (PBMC)

20 34 20 20

In vitro (PBMC) In vitro (PBMC) Serum Serum, and in vitro (PBMC) Serum In vitro (PBMC)

52 79

Results

Reference

Increased production and inhibitory effect on IL-12 Increased production Increased production in patients with anti-DNA antibodies, but diminished with respect to the controls Inhibited expression by IL-12 and IFN-␥ Increased production Increased levels Increased levels

11 12 15 19 17 10 21

Correlation with the activity of SLE Similar IL-10 secreting cells in patients and controls

22 39

PBMC, peripheral blood mononuclear cells.

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Table 7: IFN-␥ in Patients with SLE

N

Type of Study/ Sample

24 34 20 39 20 79

In vitro In vitro In vitro Serum Serum In vitro

(PBMC) (PBMC) (PBMC)

(PBMC)

Results

Reference

Positive correlation with IL-12 levels Tendency to diminish with disease activity IFN-␥ diminishes the expression of IL-10 Increased levels Correlation with IL-18 levels Lower number of IFN-␥ secreting cells in premenopausal patients than in controls

12 17 19 13 10 39

PBMC, peripheral blood mononuclear cells.

thymocytes (28). 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. Other studies have shown similar results or diminished levels (Table 8), suggesting that IL-4 does not have such a preponderant role in SLE. Despite the fact that we did not observe significant differences in cytokine levels related to treatment, it remains possible that that level may be influenced by immunosuppressive therapy (3). Glucocorticoids inhibit the production of proinflammatory cytokines, such as IL-12, TNF-␣, and IFN-␥, whereas they stimulate the production of anti-inflammatory cytokines, such as IL-10, IL-4, and transforming growth factor ␤ (41). The study on cytokines in SLE patients could be better assessed, if the genetic background of individuals were taken into account. For instance, it is known that carriers of the 8.1 ancestral haplotype

(autoimmune haplotype), which is associated with SLE but is also common in the general population, produce more TNF-␣ and IL-10 than noncarriers (42,43). 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 (44 – 47). The results obtained should enhance our understanding of the immunology of SLE and other autoimmune diseases. ACKNOWLEDGMENT The authors thank Drs. Seema S. Ahuja and Sunil Ahuja, Health Science Center at the University of Texas, San Antonio, TX, for their contributions in the development of the present work. They also express their gratitude to the patients who participated in this study and to Monica Stone for her help with the manuscript.

Table 8: IL-4 in Patients with SLE

N 20 34 20 79

Type of Study/ Sample Serum In vitro (PBMC) Peripheral blood T cells In vitro (PBMC)

Results

Reference

Increased levels and negative correlation with IL-18 levels. Decreased production Intracellular expression of IL-4 similar to that of controls and lack of correlation with disease activity. Lower number of IL-4 secreting cells in premenopausal patients than in controls

14 17 15

PBMC, peripheral blood mononuclear cells.

39

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