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Evaluation of B-cell activating factor in corticosteroid-treated systemic lupus erythematosus patients

Mashaly, Heba M.a; Helal, Hendb; Shaker, Olfat G.c

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Journal of the Egyptian Women's Dermatologic Society: July 2011 - Volume 8 - Issue 2 - p 84-89
doi: 10.1097/01.EWX.0000397888.56807.aa
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Abstract

Introduction

B-cell activating factor (BAFF) of the tumor necrosis factor (TNF) family, also known as BLyS, THANK, TALL-1, was first mentioned in the literature in 1999 [1–3] and has received increased attention as one of the key regulators of normal B cell function, tolerance, proliferation, and survival since then [4–6]. It is considered to be an immune checkpoint for B cells [7]. Over the years, B cells proved to have a more critical function in regulating immune responses than just being the precursors of antibody-secreting cells [5,8]. BAFF is considered to be a crucial homeostatic cytokine for the development of B-cell tolerance and in linking adaptive to innate immunity [8].

The role of BAFF in autoimmune B-cell induction was investigated and it could be explained by its ability to promote the survival of all B cells, even those that are autoreactive [9]. With the continuous delivery of autoantigens and increasing amounts of BAFF, the autoimmune response is aggravated, the autoreactive B cells expand and differentiate, a process that inevitably ends in the development of an autoimmune disease [7,10–12]. Other investigators thought that BAFF may play a pathogenic role in autoimmune diseases through stimulation of T-cell-dependant B-cell autoantibody production [13].

Overexpression of BAFF is considered to be a characteristic feature of systemic autoimmune diseases, particularly systemic lupus erythematosus (SLE) [14]. Expression of BAFF in the sera of SLE patients was first evaluated in 2001 by two distinct groups of investigators [10,15] who worked on two cross-sectional studies. Both studies detected significantly elevated levels of BAFF in SLE patients when compared with controls. Further studies followed and detected elevated BAFF levels in SLE patients and confirmed its crucial role in the development and progression of SLE as a well-known representative of systemic autoimmune diseases [16,17].

Corticosteroid therapy is a well-known and documented modality of treatment of SLE [18]. Studies have shown reduction of BAFF, back to its normal range, in SLE patients after their treatment with intensive short courses of high-dose steroid therapy (in the form of 60 mg prednisone orally daily) [16,19].

The effect of low-dose long-term corticosteroid therapy on BAFF level, together with clinical and laboratory findings in SLE patients, was not thoroughly investigated and needed further clarification.

This study aimed to evaluate the effect of low-dose long-term corticosteroid therapy (20 mg prednisone orally daily) on serum BAFF levels. The study also aimed at relating the BAFF level with various clinical and laboratory features detected in the included SLE patients.

Patients and methods

This study is a comparative, observational, cross-sectional study conducted on the sera of 40 Egyptian SLE patients receiving low-dose corticosteroid therapy orally daily and 40 healthy control cases. Patients were recruited from the Outpatient Clinics of the Dermatology and the Rheumatology Departments, Faculty of Medicine, Cairo University, Cairo, Egypt. All patients and apparently healthy normal controls gave informed consent.

Participants

Selection of patients

The clinical diagnosis of SLE in all patients was in accordance with the American College of Rheumatology revised criteria for SLE [20], and all selected patients were follow-up patients in remission arriving at the outpatient clinics. To be enrolled, each patient had to be receiving low-dose corticosteroid therapy in the form of 20 mg prednisone/day for a period of 6 months, without the use of any other immunosuppressive or cytotoxic drugs.

Methods

Clinical data were taken from each patient including age, sex, and disease duration, history of fever, fatigue, or weight loss. Clinical examination was conducted for each patient, on both the dermatological and the rheumatological levels.

Patients were examined for the presence of skin lesions in the form of malar rash, photosensitivity, discoid lupus erythematosus (LE)-like lesions, or oral ulcerations. They were examined by the rheumatologist for evaluation and assessment of possible arthralgia or arthritis in the form of joint swelling, hotness, or tenderness in addition to evaluation of the range of movement of each joint.

Nephritis, being a critical clinical feature in patients with SLE, was also evaluated by measuring 24 h urinary proteins, and it was diagnosed by the presence of persistent proteinuria greater than 0.5 g/day or greater than three plus signs if quantization was not performed.

A blood sample was taken from each patient for serological workup, including evaluation of first-hour and second-hour erythrocyte sedimentation rate (ESR) levels, and complete blood picture. Serological evaluation also included assessment of antinuclear antibodies (ANAs), antidouble stranded DNA (anti-dsDNA) antibodies, and evaluation of BAFF level. A blood sample was taken from each control case to measure only the BAFF level.

Measurement of BAFF level using ELISA

Serum BAFF levels were measured using a specific enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, Minnesota, USA) according to the manufacturer's protocol. The detection limit of this assay was 0.00338 ng/ml. This assay uses the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for BAFF was precoated onto a microplate. Standards and samples were pipetted into the wells, and any BAFF that was present was bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for BAFF is added to the wells. After a wash, to remove any unbound antibody–enzyme reagent, a substrate solution is added to the wells, and color develops in proportion to the amount of BAFF bound in the initial step. The color development is stopped and the intensity of the color is measured using enzyme-linked immunosorbent assay reader. The concentration of the BAFF in serum samples is calculated after creation of the standard curve.

Statistical analysis

Data were statistically described in terms of range, mean±standard deviation, median for quantitative data, and frequencies (number of cases) and percentages for qualitative data. Comparison of quantitative variables between the study groups was made using Mann–Whitney U-test for independent samples. For comparing categorical data, χ2-test was carried out. Fisher's exact test was used instead when the expected frequency was less than 5. A probability value (P value) of less than 0.05 was considered to be statistically significant. All statistical calculations were made using computer programs such as Microsoft Excel 2003 (Microsoft Corporation, New York, USA) and Statistical Package for the Social Science (SPSS Inc., Chicago, Illinois, USA) version 15 for Microsoft Windows.

Results

The study included 40 SLE patients receiving corticosteroids orally daily in a dose of 20 mg/day for a period of 6 months. The patients included 32 females (80%) and eight males (20%); their age ranged from 17 to 52 years, with a mean of 29.20±8.5 years. Disease duration ranged from 1 to 9 years, with a mean of 7.18±4.55 years. The study also included 40 apparently healthy participants, 31 women and nine men; their age ranged from 20 to 54 years, with a mean of 30.12±8 years.

With regard to general constitutional manifestations, 22 out of the 40 (55%) included SLE patients developed fever, 14 patients (35%) complained of constant fatigue, and 13 patients (32.5%) reported a history of weight loss.

Skin lesions were present in 32 (80%) SLE patients, 29 patients of who had malar rash whereas only three patients had discoid LE-like lesions. History of photosensitivity was present in 35 out of 40 (87.5%) SLE patients.

Rheumatological examination revealed the presence of arthritis in 32 out of 40 (80%) patients. Nephrological involvement, presented by persistent proteinuria, was detected in 24 out of the 40 included SLE cases (60% of patients).

Elevated ESR was detected in 34 patients (85%), whereas pancytopenia was found in 10 patients (25%). Positive ANA was detected in almost all included SLE patients except for two patients (38 patients, 95%). Positive anti-dsDNA antibodies were found in 33 included SLE cases (82.5% of patients). Summary of all the patients' clinical and laboratory data is illustrated in Table 1.

Table 1
Table 1:
Summary of general, dermatological, rheumatological, and laboratory evaluation of SLE patients

Estimation of BAFF level in SLE patients

The level of BAFF in the sera of SLE patients was considered to be elevated when it was higher than its mean level in the controls (0.517±0.175 ng/ml). Elevated BAFF level was detected in 30 patients (75% of patients) with a mean level of 1.468±1.540 ng/ml (it ranged from 0.8 to 6 ng/ml). The difference between the mean BAFF level in SLE patients and controls was found to be statistically highly significant, with a P value of less than 0.001 (Table 2), indicating the significant elevation of BAFF level in SLE patients receiving low-dose steroid therapy when compared with healthy controls.

Table 2
Table 2:
Statistical comparison between mean BAFF level in controls and treated SLE patients

Statistical relationship between elevated BAFF level and other included variables

Statistical relationship between elevated BAFF level and other clinical and laboratory findings was done and it proved to be statistically nonsignificant as regards age, fever, fatigue, weight loss, malar rash and discoid LE-like lesions, photosensitivity, arthritis, proteinuria, elevated ESR, and pancytopenia. The statistical relationship between elevated BAFF level and positive expression of either ANA or anti-dsDNA antibodies also proved to be nonsignificant. Further details of the tests of significance and P values are mentioned in Tables 3 and 4.

Table 3
Table 3:
Statistical relationships between BAFF level (normal or elevated) and different categorical clinical and laboratory data in the 40 corticosteroid-treated SLE cases using Pearson's χ2-tests
Table 4
Table 4:
Statistical relationships between BAFF level (normal or elevated) and different quantitative clinical data in the 40 corticosteroid-treated SLE cases using Mann–WhitneyU-test

The only variable that showed a significant statistical relationship with elevated BAFF level was disease duration (P value=0.005), in which the longer the disease duration, the higher the BAFF level.

Discussion

This study aimed to evaluate the effect of low-dose long-term daily steroid regimen on the changes of BAFF level. Significantly elevated BAFF level was detected in 75% of our included SLE cases receiving daily oral steroids in a dose of 20 mg prednisone for a period of 6 months. On reviewing the literature for the effect of steroids on BAFF level, we encountered a study demonstrating elevated BAFF level in 20% of 95 SLE patients in remission receiving prednisone with a mean dose of 18.8 mg orally daily [10], a finding that was very similar to our finding in spite of the higher percentage of elevated BAFF level in this study. On further perusing the issue, we found a study that reported that treatment of eight SLE patients with high-dose corticosteroids (60–80 mg prednisone orally daily) resulted in marked reduction in the serum BAFF level down to its normal range. Tapering of corticosteroids to 30–40 mg/day resulted in a rebound increase of serum BAFF level [16]. In addition, a Chinese study [19] denoted marked reduction of BAFF after a week of high-dose glucocorticoid therapy in patients with SLE and lupus nephritis. Another nondermatological study demonstrated lowering of the BAFF level to normal in 2 weeks after low-dose steroid in a dose of 40 mg prednisone daily for 4 consecutive days in 20 patients with immune thrombocytopenic purpura [21] and rheumatoid arthritis [22]. It seems that low corticosteroids are ineffective in normalizing serum level of BAFF, and for this to happen, it needs a relatively high dose of corticosteroids of 40 mg prednisone or more.

It seems that the same concept of lack of effectiveness of low steroid doses (lower than 40 mg/day) may imply for clinical and serological findings, as considerable percentages of our included SLE patients were still exhibiting constitutional manifestations in the form of fever (55%), fatigue (35%), and weight loss (32.5%). They were still showing skin lesions in the form of malar rash and discoid LE-like lesions (80%), photosensitivity (87.5%), arthritis (80%), renal involvement (60%), elevated ESR (85%), and pancytopenia (25%).

In this study, elevated BAFF level showed no statistically significant relationship with age or sex of patients. In contrast, a significant statistical relationship was detected between elevated BAFF level and disease duration, indicating that the longer the disease duration, the higher the BAFF level. On reviewing the literature, we found only one study tackling the disease duration and showing an inverse relationship between bullous pemphigoid disease duration and BAFF level in which elevated BAFF levels preceded the development of the full criteria of bullous pemphigoid, indicating that BAFF may be a useful marker for early activation of an autoimmune diathesis [23].

The results of our study showed no significant relationship between elevated BAFF level and the presence of malar rash, discoid LE-like lesions, or arthritis. No statistical relationship was detected between elevated BAFF level and elevated ESR. This last finding is supported by Zhang et al. [15] who denoted a lack of correlation between elevated BAFF level and elevated ESR as a marker of disease activity and used this finding to conclude that elevated BAFF level seems to be an unlikely marker for activity or severity of SLE. Stohl et al. [16] noted that lack of association between elevated serum BAFF level and organ involvement is not a surprising finding, as BAFF has no known direct or immediate proinflammtory properties. Therefore, elevated or reduced BAFF levels should not be expected to promote increase or decrease in systemic or organ-specific inflammation.

As for renal involvement, manifested by proteinuria, we were not able to find any statistical relationship between elevated BAFF level and proteinuria in our treated SLE cases. A previous study [10] has demonstrated an inverse relationship between persistent proteinuria and BAFF level in which proteinuria is significantly associated with low BAFF levels, and they explained it by the fact of concomitant loss of BAFF in urine due to heavy proteinuria. Wang et al. [19] reported significant correlation between elevated BAFF level and grade IV lupus nephritis, indicating that the higher the BAFF level, the more severe the grade of lupus nephritis, a finding that we could not prove or confirm in our study.

Statistical relationship between elevated BAFF level and autoantibodies was investigated in this study and also in previous studies. The results of this study could not find a significant relationship between elevated BAFF level and positive expression of neither ANA nor anti-dsDNA antibodies. This comes in accordance to a study conducted by Becker Merok et al. [24], which was also unable to find a relationship between elevated BAFF level and anti-dsDNA antibodies. However, Cheema et al. [10], Chu et al. [12], and Lin et al. [25] demonstrated positive correlation between elevated BAFF level and titer of anti-dsDNA antibodies. Zhang et al. [15] demonstrated that the percentage of positive anti-dsDNA antibodies was significantly higher in patients with elevated BAFF level in SLE patients. The lack of a significant relationship between elevated BAFF level and the level of autoantibodies detected in this and in the study conducted by Becker Merok et al. [24] may indicate that BAFF stimulation of B cells may contribute to SLE by mechanisms other than autoantibody production.

Tempted by the critical role of BAFF in SLE disease pathology, investigators evaluated the possibility of BAFF-blocking agents as new emerging steroid-sparing therapeutic modalities in the treatment of SLE [21]. Borrowing from the successful clinical experience with tumor necrosis factor antagonism [11], a phase I clinical trial of SLE with a fully human antihuman BAFF monoclonal antibody was initiated in early 2002 and the results of the trial has shown that it is safe and biologically effective [26]. Phase II studies of SLE have recently been completed with a blocking antibody to BAFF, termed belimumab, which demonstrated a high level of safety and some efficacy [27,28]. Additional antibody-based and receptor-based antagonists against BAFF are still in development [8,29,30], and being highly selective of its cellular targets (B cells), they have a chance in becoming important and valuable steroid-sparing therapeutic modalities in the future of treatment of SLE.

As a conclusion, it seems that the oral steroid dose of 20 mg/day is ineffective in lowering the BAFF level to its normal level and could not bring clinical and laboratory findings in our SLE patients under control. Although BAFF was previously mentioned by previous studies as a key factor in the pathogenesis of SLE, yet, we could not find any correlation between elevated BAFF level and cutaneous, rheumatological, renal, or laboratory findings in our treated SLE patients. We can, then, postulate that multiple factors and cytokines contribute to the pathogenesis and disease activity of SLE, and BAFF is only one of them.

Future studies can be directed to evaluate changes in BAFF level and SLE disease activity when low-dose steroids are combined with other immunosuppressive drugs or antimalarials.

There is no conflict of interest to declare.

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Keywords:

B-cell activating factor; corticosteroids; systemic lupus erythematosus

© 2011 Egyptian Women's Dermatologic Society