Vitamin D status in systemic lupus erythematosus patients and its association with selected clinical and laboratory parameters

Abstract

Objectives: To identify relationships between vitamin D serum levels and the presence of autoantibodies directed against vitamin D and levels of interleukin(IL)-17 and IL-23 in patients with systemic lupus erythematosus (SLE). Methods: The study included 49 patients with SLE. Serum concentrations of 25(OH)D₃ were measured with electrochemiluminescence immunoassay (ECLIA). Enzyme-linked immunosorbent assays (ELISA) were used to determine antibodies directed against 1,25(OH)₂D₃ and levels of IL-17 and IL-23 in serum of SLE patients. In evaluation of vitamin D status, the control group consisted of 49 age and gender matched healthy individuals, whereas in assessment of anti-vitamin D antibodies the control group comprised 30 sera from blood donors. Results: Serum concentration of 25(OH)D₃ in SLE patients during the warm season was 18.47 ± 9.14 ng/ml, which was significantly decreased as compared with that of the control group – 31.27 ± 12.65 ng/ml (p = 0.0005). During the cold season a trend toward lower concentration of 25(OH)D₃ in SLE patients was revealed; however, it did not reach statistical significance (11.71 ± 7.21 ng/ml vs. 16.01 ± 8.46 ng/ml; p = 0.054). Results within the recommended range for vitamin D (30–80 ng/ml; 70–200 nmol/l) were observed only in three patients. The 25(OH)D₃ concentration was decreased in SLE patients with renal disease or leucopenia as compared with the levels in patients who did not have either problem (p = 0.006 and p = 0.047, respectively). The cold season was found to be a risk factor for vitamin D deficiency (<20 ng/ml) (odds ratio = 9.25; p = 0.005). Autoantibodies directed against 1,25(OH)₂D₃ were detected in three SLE patients. No significant difference in 25(OH)D₃ serum concentrations was found between SLE patients with and without these autoantibodies. No link was shown between the existence of autoantibodies against 1,25(OH)₂D₃ and clinical or laboratory findings, including IL-17 and IL-23 levels. However, serum concentrations of IL-23 were lower in patients with vitamin D deficiency (p = 0.037). Conclusions: SLE patients, especially those with leucopenia or renal involvement, are at high risk of vitamin D deficiency and require vitamin D supplementation. Some SLE patient sera contained 1,25(OH)₂D₃ antibodies, but these antibodies do not appear to affect vitamin D levels.

Keywords

hematologic changes renal lupus systemic lupus erythematosus

Introduction

Systemic lupus erythematosus (SLE) is an autoimmune disease that affects connective tissue and blood vessels in the skin and internal organs.¹ The pathogenesis of SLE is multifactorial, and includes genetic, hormonal, immunopathologic, and environmental factors.¹ Most patients are photosensitive to ultraviolet radiation (UVR).² UV exposure may not only cause an exacerbation of skin lesions but may also aggravate systemic features and induce the production of anti-double-stranded DNA (anti-dsDNA) autoantibodies.² Therefore patients are routinely advised to take sun-protective measures that include avoiding sun exposure by wearing tightly woven clothes and broad brimmed hats and by using sunscreens.³ Although photo-protective measures are important in treating SLE patients, these measures may result in vitamin D insufficiency⁴ by blocking UVB-induced synthesis of vitamin D₃ (cholecalciferol) in the skin.⁵ Vitamin D plays a crucial role in calcium–phosphorus homeostasis and influences immune modulation and cellular differentiation and growth. Hence Vitamin D deficiency may precipitate adverse effects on many tissues and organs^6,7 and contributes to osteopenia and osteoporosis, conditions that are found frequently in SLE, including up to 68% of SLE patients undergoing systemic glucocorticoid therapy.^8,9 On the other hand, it is also possible that autoantibodies directed against vitamin D in serum of SLE patients may inhibit the effects of vitamin D.¹⁰ In addition, when considering a role of vitamin D in autoimmune diseases, one should take into account immunomodulatory properties of vitamin D, such as inhibition of antigen-induced T-cell activation and subsequent blastic transformation, dendritic cell differentiation and maturation, production of interleukin (IL)-2, IL-6, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, and augmentation of IL-4, IL-5, IL-10, and transforming growth factor (TGF)-β1 expression.⁶ Therefore, it is conceivable that vitamin D deficiency participates in immunological dysregulation. It would appear that in SLE there is an imbalance between T and B lymphocytes, with impaired cellular immunity and hyperactive humoral antibody response, possibly connected with a relative deficiency of suppressor and/or inducer T cells.¹ In light of the fact that the IL-23/IL-17 axis is a significant mediator of inflammation, levels of both IL-17 and IL-23 in SLE patients were evaluated. There is little information in the medical literature about the relationships between serum vitamin D levels, the presence of anti-vitamin D antibodies, and serum levels of IL-17 and IL-23 in SLE patients. For that reason the aim of the study was to evaluate the vitamin D status in patients with systemic lupus erythematosus and its association with clinical and laboratory parameters, including autoantibodies directed against vitamin D and levels of IL-17 and IL-23.

Patients and methods

The study included a limited number of 49 patients with SLE, including 47 females and two males, between the ages of 20 and 67, mean 43.71 ± 11.51 years. The diagnosis of SLE was based on classification criteria for SLE updated in 1997 by the American College of Rheumatology.¹¹ Data were collected from anamnesis or documents in medical records. The duration of SLE ranged from 0.5 to 31 years, mean 6.98 ± 7.23 years. The mean Systemic Lupus Activity Measure (SLAM) score ranged from 2 to 34, mean 10.57 ± 5.95.¹² Height and weight of the patients were estimated and then BMI was computed. Usage of sunscreen with protection factor >15, applied every time before going outdoors, was assessed in SLE patients. Subjects who had used antiepileptics, anticoagulants or vitamin D supplementation within 6 months prior to enrollment were excluded from the study. In evaluation of vitamin D status, the control group consisted of 49 age and gender matched healthy individuals, whereas in assessment of anti-vitamin D antibodies the control group comprised 30 sera from healthy blood donors. All SLE patients and controls were Caucasians with skin phototype II or III according to the Fitzpatrick classification.¹³ All participants gave their informed consent prior to the study. The project was approved by the local Ethics Committee (No RNN/67/08/KE). As the study population was located in the northern hemisphere, in the estimation of seasonal variation of vitamin D status the cold season was defined as the period between 5 October and 5 April, while the warm season comprised the remaining months.

Evaluation of 25(OH)D₃ concentration

Serum concentration of 25(OH)D₃ was measured with reagents including calibrations and control sera obtained from Roche Diagnostic, Mannheim, Germany (Catalog number: 11706802001, 11706799001, 11776576322, 10394246001, 03314847190) with electrochemiluminescence immunoassay (ECLIA) in the automated analyzer Elecsys 2010 (Roche Diagnostic, Mannheim, Germany).

Both inter-assay and intra-assay variations were <15%. 25(OH)D determinations were under international control of the Vitamin D External Quality Assessment Scheme (DEQAS) with Certificate of Proficiency. Vitamin D status was based on the results of serum concentration of 25(OH)D₃ and defined as follows: deficiency <20 ng/ml (<50 nmol/l), insufficiency 21–29 ng/ml (52.5–72.5 nmol/l) and recommended range 30–80 ng/ml (72.5–200 nmol/l).¹⁴^–¹⁶.

Detection of antibodies directed against 1,25(OH)₂D₃

In order to determine the presence of antibodies directed against 1,25(OH)₂D₃ in serum of 40 patients, enzyme-linked immunosorbent assay (ELISA) was employed according to Carvalho et al. with several modifications.¹⁰ A solution of 1,25(OH)₂D₃ (Pharmaceutical Institute, Poland) at a concentration of 5 µg/ml in 70% ethanol was pre-coated onto a microplate (Maxisorb, Nunc, Denmark). Than the plate was blocked with bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Serum samples diluted 1:100 with BSA were pipetted into the wells and incubated for 4 h at room temperature. After any unbound substances had been washed away, 50 μl of polyvalent anti-human immunoglobulin conjugate was added to each well. After a wash that removed any unbound antibody–enzyme reagent, a substrate solution was added to the wells and color developed in proportion to the amount of autoantibodies directed against 1,25(OH)₂D₃. The color development was stopped and the intensity of the color was measured. Each serum sample was measured in triplicate. Results more than 3 standard deviations (SD) above the optical density obtained in serum samples from healthy control individuals were considered to be positive.

Measurement of interleukin (IL)-17 and IL-23

The quantitative determination of IL-17 was performed using the Quantikine immunoassay (R&D Systems Cat No D1700), according to the manufacturer’s protocol. Recombinant human IL-17 (from R&D Systems) and IL-23 (NatuTec) were used as the standards. Optical density was measured using an automatic ELISA reader (LP400; Diagnostics Pasteur, Marnes-La-Coquette, France). The detection limit was 15 pg/ml for IL-17 and 156 pg/ml for IL-23.

The ELISA for human IL-23 was performed using antibodies specific for particular subunits of this cytokine: rat IgG1 anti-p19 IL-23 subunit as the capture antibody and biotinylated mouse IgG1 anti-p40/p70 IL-12 subunits as the detection antibody (both from NatuTec, Germany; cat. no. 14-7238 and 13-7129, respectively). Then streptavidin–horseradish peroxidase conjugate (Jackson ImmunoResearch, West Grove, PA), followed by o-phenylenediamine dihydrochloride, was utilized to develop the enzymatic reaction.

Statistics

Statistical analysis was performed using Statistica software version 6.0 (Statsoft, Poland). Data estimation with the Shapiro–Wilk test did not confirm a Gaussian distribution. Therefore the non-parametric Mann–Whitney U test was employed to compare interval variables between two groups. Fisher’s exact test was used to assess association between dichotomous variables, and Spearman’s rank test to assess correlation. In order to find the best predictor of vitamin D status among such variables as age, gender, BMI, and disease duration, multiple regression analysis was performed. Logistic regression analysis was employed in order to estimate odds ratio for seasons of the year as a risk factor for vitamin D deficiency.

Results

Clinical characteristics of the studied group of patients, according to revised ACR criteria for classification of SLE, are shown in Table 1.

Table 1

Clinical characteristics of SLE patients according to frequency

Criterion		Number of individuals (Percentage)
ANA presence (titer >1:160)		49 (100%)
Arthritis/arthralgia		46 (93.87%)
Photosensitivity		42 (85.71%)
Malar rash		41 (83.67%)
Hematologic disorder	Leukopenia	32 (65.3%)
	Hemolytic anemia	22 (44.89%)
	Thrombocytopenia	18 (36.73%)
	Lymphopenia	17 (34.69%)
Oral ulcers		19 (38.77%)
Renal disorder		18 (36.73%)
Immunologic disorder	Anticardiolipin antibodies IgG or IgM	14 (28.57%)
	Anti-dsDNA	9 (18.36%)
	Anti-Sm	7 (14.28%)
	Lupus anticoagulant	4 (8.1%)
	VDRL – false positive	1 (2.04%)
Discoid rash		11 (22.44%)
Neuropsychiatric disorder		6 (12.24%)
Serositis		2 (4.08%)

ANA: antinuclear antibody, dsDNA: double-stranded DNA, SLE: systemic lupus erythematosus, VDRL: Venereal Research Disease Laboratory.

Serum concentration of 25(OH)D₃ in patients with SLE during the warm season was 18.47 ± 9.14 ng/ml, which was significantly decreased as compared with that of the control group – 31.27 ± 12.65 ng/ml (p = 0.0005) (Figure 1). During the cold season a trend toward lower concentration of 25(OH)D₃ in SLE patients was revealed; however, it did not reach statistical significance in comparison with the control group (11.71 ± 7.21 ng/ml vs. 16.01 ± 8.46 ng/ml, respectively; p = 0.054) (Figure 2).

Vitamin D status assessed separately during the warm season and the cold season in SLE patients and controls is shown in Table 2.

Table 2

Vitamin D status in SLE patients and controls

Studied group (Number of individuals)	Season	Vitamin D status (25(OH)D₃ concentration)
Studied group (Number of individuals)	Season	Deficiency (<20 ng/ml; <50 nmol/l)	Insufficiency (21–29 ng/ml; 52.5–72.5 nmol/l)	Recommended (30–80 ng/ml; 72.5–200 nmol/l)
SLE N = 49	Warm season N = 22/49	11/22 (50%)	9/22 (40.9%)	2/22 (9.09%)
SLE N = 49	Cold season N = 27/49	24/27 (88.8%)	2/27 (7.4%)	1/27 (3.7%)
Control group N = 49	Warm season N = 27/49	4/27 (14.8%)	11/27 (40.7%)	12/27 (44.44%)
Control group N = 49	Cold season N = 22/49	16/22 (72.7%)	6/22 (27.27%)	0/22

SLE: systemic lupus erythematosus.

25(OH)D₃ concentration was significantly decreased in SLE patients with renal disease and leucopenia in anamnesis as compared with the levels in patients who did not fulfill these criteria (p = 0.006 and p = 0.047, respectively) (Figures 3 and 4).

We did not find any significant association between the presence of anti-dsDNA antibodies and 25(OH)D₃ concentration, between 25(OH)D₃ concentration and SLAM score, or between serum 25(OH) vitamin D₃ concentration and age, gender, and disease duration. BMI was not correlated with 25(OH)D₃ in SLE patients. No significant difference was found in 25(OH)D₃ concentration between patients with BMI >30 kg/m² (obesity) and those with BMI <30 kg/m² (16.64 ± 10.93 vs. 14.08 ± 8.11). We found no significant difference in 25(OH)D₃ level between patients who were treated with glucocorticoids or antimalarials and those who were not.

The cold season of the year, not surprisingly, was found to be a risk factor for vitamin D deficiency (<20 ng/ml) (odds ratio [OR] = 9.25; p = 0.005).

Autoantibodies directed against 1,25(OH)₂D₃ were detected in three patients with SLE. No difference in 25(OH)D₃ concentration was found between SLE patients with and without autoantibodies directed against 1,25(OH)₂D₃. We did not prove any significant link between the existence of autoantibodies directed against 1,25(OH)₂D₃ and clinical or laboratory findings, including IL-17 and IL-23.

In SLE patients serum concentrations of IL-17 and IL-23 were 71.83 ± 138.19 pg/ml and 531.67 ± 890.31 pg/ml, respectively.

No significant association was observed between IL-17 and vitamin D status.

Serum concentration of IL-23 was significantly decreased in patients with vitamin D deficiency (p = 0.037).

IL-23 concentration was significantly decreased in SLE patients with renal disease and lymphopenia (p = 0.044 and 0.02, respectively).

There was no association between SLAM score and IL-17 or IL-23 concentration.

No significant correlation was found between 25(OH)D₃ and IL-17 or IL-23.

Discussion

This is the first study to address the connection between vitamin D status, anti-vitamin D autoantibodies, IL-17 and IL-23 levels in SLE. The serum concentration of 25(OH)D₃ during the warm season was lower in SLE patients than in controls. It is worth emphasizing that most patients were in a vitamin D deficiency state (25(OH)D₃ <20 ng/ml). Although 25(OH)D₃ is not a biologically active metabolite of vitamin D, it is considered as the best parameter to estimate vitamin D status for several reasons.^15,16 First of all, the concentration of 25(OH)D₃ in either serum or plasma is 1000-fold higher than that of 1,25(OH)₂D₃.¹⁷ The normal range of 25(OH)D₃ levels is 30–80 ng/ml, whereas concentration of 1,25(OH)₂D₃, depending on the method of assay, is 20–60 pg/ml.^15,16 Moreover, the half-life for 25(OH)D₃ is approximately 14 days, compared with 4–6 h for 1,25(OH)₂D₃.¹⁷ Finally, when a patient is vitamin D deficient there is often a normal or even elevated blood level of 1,25(OH)₂D₃, the biologically active metabolite of vitamin D, as a consequence of development of secondary hyperparathyroidism, increased secretion of parathormone and subsequent overexpression of the kidney’s 1 -α hydroxylase, which produces more 1,25(OH)₂D₃.¹⁷

We found vitamin D₃ deficiency in most of the SLE patients. Results within the normal range were observed in only three (6.12%) patients. Our findings corroborate those of Thudi et al., whose study conducted on 37 women with SLE demonstrated vitamin D deficiency, defined as serum 25(OH)D₃ level <47.7 nmol/l), in 20% of patients.¹⁸ Moreover, these results are consistent with those of Orbach et al., whose study revealed that mean serum 25(OH)D₃ concentration was 11.9 ± 11.1 ng/ml.¹⁹ Kamen et al. found a trend toward reduced 25(OH)D₃ levels in SLE patients compared with controls.²⁰ Critically low vitamin D levels (<10 ng/ml) were found in 22 of the SLE cases, with presence of renal disease being the strongest predictor (OR 13.3, p < 0.01), followed by photosensitivity (OR 12.9, p < 0.01).²⁰ In accordance with these data, in our group of Caucasian patients 25(OH)D₃ concentration was significantly decreased in SLE patients with renal disease. Moreover, the cold season of the year was found to be a risk factor for vitamin D deficiency.

In our study we have found autoantibodies directed against 1,25(OH)₂D₃ at low frequency. In the face of a wide spectrum of autoantibodies detected in SLE, currently including at least 116 autoantibodies, the presence of antibodies directed against 1,25(OH)₂D₃ identifies another autoantibody in SLE.²¹ However, they do not appear to have a significant effect on vitamin D status, at least as measured by 25(OH)D concentration. Nevertheless, it is conceivable that the antibodies counteract the biological effects of calcitriol in vivo or the precision of measurement of vitamin D metabolites in vitro. These suspicions need further evaluation. In the first, and to our knowledge the only, study on antibodies against 1,25(OH)₂D₃ in SLE, conducted by Carvalho et al., autoantibodies of isotype G were detected in 7% of SLE patients.¹⁰ In addition, titers of anti-dsDNA antibodies were higher in patients with antibodies directed against 1,25(OH)₂D₃.¹⁰ On the contrary, our study did not reveal any clinical association between these antibodies and the course of SLE; however, further study is merited. As there is no simple explanation for how low vitamin D status influences autoimmunity in SLE, one should realize that vitamin D deficiency has many nonskeletal consequences. There is evidence that vitamin D deficiency is associated with hypertension, increased risk of coronary artery disease, congestive heart failure, and development of autoimmune disorders.¹⁷ However, the explanation for decreased vitamin D status in patients with leukopenia requires further investigation. Our attempt to resolve the problem has focused on IL-17 and IL-23. Also, there is a lack of data on IL-17 and IL-23 levels in SLE patients.

IL-17 is a proinflammatory cytokine formerly identified as playing a defensive role against microorganisms and a pathogenic role in autoimmune diseases.²² In 2008, for the first time, Wong et al. found an increased level of IL-17 in serum of patients with SLE.²³ This molecule was initially known as mouse cytotoxic T lymphocyte-associated antigen-8 (CTLA-8).²⁴ CD4+, CD8+, invariant NK and γδ T cells, neutrophils, monocytes, and eosinophils are capable of synthesizing IL-17.²⁵^–²⁹ The role of IL-17 seems to be connected with communication between the immune system and the hematopoietic system, because IL-17, derived from T cells, induces fibroblasts to synthesize IL-6, IL-8, ICAM-1 and G-CSF.³⁰ In addition, IL-17 enhances proliferation of partially activated T cells and upregulates nitric oxide production in osteoarthritic cartilage.^30,31

In turn, the source of IL-23 is restricted to dendritic cells and macrophages. This molecule is synthesized in response to pathogens, such as bacteria and viruses.³² At the mRNA level IL-23 was found to be elevated in SLE patients.³³ IL-23 plays a role in the development/maintenance of a T-cell subset characterized by the synthesis of IL-17 A, IL-17 F, IL-6, and TNF-α and induces the initial recruitment of neutrophils to the site of infection.³³^–³⁵ It seems that the effects of IL-23 on IL-17-producing T cells may also augment the development of numerous models of disease with autoimmunologic pathogenesis, such as diabetes, colitis, experimental allergic encephalomyelitis, and collagen-induced arthritis.³⁵^–⁴¹

In light of the fact that the IL-23/IL-17 axis is a significant mediator of inflammation, in our study levels of both IL-17 and IL-23 were evaluated in SLE patients. We did not find any correlation between IL-17 and IL-23 levels. Serum concentration of IL-23 was significantly decreased in patients with vitamin D deficiency (<20 ng/ml). However, the molecular basis underlying this finding remains unidentified. Surprisingly, IL-23 concentration was significantly decreased in SLE patients with renal disease and lymphopenia (p = 0.044 and 0.02, respectively). At present, it is uncertain whether the co-occurrence of decreased IL-23 level, renal disorder, and lymphopenia is the result of a cause-and-effect relationship or is only a coincidence. The clinical interpretation of analysis of cytokines in SLE, such as IL-17, and IL-23 in general, is complicated because of variable results derived from studying serum levels, secretion into culture supernatants, and analysis of intracellular mRNA or proteins.⁴² There are a limited number of studies addressing the role of IL-23/IL-17 in renal involvement in SLE and no reports of its relation with vitamin D. An aberrantly active IL-23/IL-17 axis was found to contribute to the development of nephritis in lupus-prone mice. Lymphocytes isolated from lupus-prone animals progressively express higher levels of IL-23 receptor mRNA as their disease becomes more severe.⁴² On the other hand, inhibited secretion of IL-23 in the regulatory function of vitamin D treated dendritic cells was revealed.⁴³ Because of lack of comprehensive data regarding IL-23 and vitamin D in SLE, further investigations are required to elucidate their roles in inflammation and the course of disease.

In contrast to the limited number of studies of IL-17 and IL-23, studies of IL-6, IL-10, and IFN-α have shown increased levels and clinically relevant observations in most SLE patients.⁴⁴ Although aberrant immune function seems to result from intrinsic immunologic abnormalities, hormonal influences, and environmental triggers, cytokine network imbalance seems to play a contributory role. Hence, molecular studies determining the role of IL-23 and vitamin D in immune cells are required to enhance knowledge of their participation in the pathogenesis of SLE.

The results from this study indicate that SLE patients are at a high risk of vitamin D deficiency and that they should receive vitamin D supplementation. It also seems particularly prudent to provide supplementation to SLE patients with renal disease and leucopenia, based on our findings. Future studies are needed to further explore the role that vitamin D deficiency might play in SLE and other disorders.

Footnotes

Funding

This work was supported by the Medical University of Lodz, Poland, grant no. 503-1019-1.

Conflict of interest

None declared.

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Vitamin D status in systemic lupus erythematosus patients and its association with selected clinical and laboratory parameters

Abstract

Keywords

Introduction

Patients and methods

Evaluation of 25(OH)D3 concentration

Detection of antibodies directed against 1,25(OH)2D3

Measurement of interleukin (IL)-17 and IL-23

Statistics

Results

Discussion

Footnotes

Funding

Conflict of interest

References

Evaluation of 25(OH)D₃ concentration

Detection of antibodies directed against 1,25(OH)₂D₃