Effects of tetra-arsenic tetra-sulfide on BXSB lupus-prone mice: a pilot study

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disease of uncertain etiology that affects multiple tissues and organs. Arsenic trioxide (ATO) has been used in lupus-prone mice with a regulatory effect on immune abnormality. Tetra-arsenic tetra-sulfide (As₄S₄), a traditional Chinese medicine, is effective on acute promyelocytic leukemia with mild side effects than ATO. In this study, a pilot study was performed to investigate the effects and the mechanism of As₄S₄ on the lupus-prone BXSB mice. Improvement of monocytosis (p < 0.05) in spleen and decreased serum interleukin-6 (IL-6) (p = 0.0277) were observed with As₄S₄ treatment. As₄S₄-treated mice exhibited amelioration of skin, liver and renal disease with mild side effects. Histological analysis revealed that As₄S₄ suppressed immune complex deposition, mesangial proliferation and inflammatory cell infiltration in kidney and liver. Our study support that As₄S₄ selectively suppresses cutaneous lupus and nephritis in BXSB mice and might be a potential treatment for SLE.

Keywords

Tetra-arsenic tetra-sulfide systemic lupus erythematosus BXSB mice

Introduction

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a variety of autoantibodies and immune complex (IC) deposition in tissues with multisystemic injuries.¹ In the BXSB-strain mouse, a lupus-prone animal, there are iterations of lupus-like symptoms akin to human SLE, including autoantibody production, lymphoid activation and hyperplasia, and some unique histological and serological manifestations were also observed with unique disease accelerators.² The disease process is strikingly accelerated in male BXSB mice with the Y-accelerating autoimmune gene (Yaa) gene.³

Standard therapy for SLE with glucocorticoids combined with immunosuppressive agents exhibits various side-effects and disease relapses after therapy discontinuation or tapered doses.⁴ Tetra-arsenic tetra-sulfide (As₄S₄) has long been used for treatment of leukemia in China. Previous studies exhibited good outcomes in the treatment of both newly diagnosed acute promyelocytic leukemia (APL) and relapsed/refractory promyelocytic leukemia with As₄S₄ mild toxicity profiles.⁵ The mechanism involved induction of cell apoptosis.⁶ To investigate efficacy and side effects, BXSB mice were treated with As₄S₄ and the clinical effect and immune parameters were studied.

Materials and methods

Mice

Male BXSB (H-2^b) mice were obtained from the Jackson Laboratory (Bar Harbor, Maine, USA, stock 000740). Male C57BL/6 (H-2^b) mice were obtained from Unilihua Bioscience Center (Beijing, China). The 28-week-old mice were housed under specific pathogen-free conditions and fed standard rodent chow and water ad libitum. The studies were approved by the Animal Care and Use Committee of Peking University People's Hospital.

Preparation of As₄S₄ and treatment of animals

As₄S₄ was kindly provided by Daopei Lu and dissolved in 0.5% methyl cellulose (Sigma-Aldrich, St Louis, Missouri, USA) for the study. As in the Lu et al. study,⁵ BXSB mice (n = 10) were treated with As₄S₄ 1000 µg/g body weight by oral gavage daily for eight weeks: the control mice (n = 10) received the same volume of 0.5% methyl cellulose. C57BL/6 mice (n = 5) received 2000 µg/g of As₄S₄ daily for eight weeks to test toxicity.

Arsenic levels in blood, hair and the complete blood cells count

At scheduled time-points (0, 1, 7, 20 and 60 days after treatment), heparinized blood and hair samples were collected. Quantitative determination of arsenic levels in blood and hair samples was performed as in the previous literature.⁵ Complete blood cell counts were analyzed with Blood Analyzer (MEK-6318K, Japan) at the end of treatment.

Urinary albumin/creatinine concentration ratios

Mice were placed in metabolic cages for 24 h urine collection. Urinary albumin was determined by use of the Mouse Albumin ELISA Kit (ICL, Newberg, USA) according to the instructions. Urinary creatinine concentrations were detected with a Beckman Auto analyzer (Beckman Coulter, Fullerton, California, USA). Urinary albumin was normalized to creatinine excretion and presented as micrograms of albumin per micromole of creatinine.

Histopathology and immunofluorescence assay

Kidney, liver, lung, ear tissue samples were collected at the time of harvest and fixed in 10% buffered formalin, embedded in paraffin. Hematoxylin and eosin (H&E) or periodic acid-Schiff-methenamine (PASM) stain were formed. For immunofluorescence staining, the kidney tissues were snap-frozen in liquid nitrogen and placed in optimum cutting temperature (OCT) compound (Sakura Japan, Osaka, Japan). Frozen sections (5 µm thick) were stained with fluorescein-conjugated anti-mouse IgG (1:50, AbD serotec, Oxford, UK) or anti-mouse IgM and C3 (1:60 and 1:30, respectively, Cedarlane, Burlington, Canada) and were visualized with a fluorescence microscope.

Lymphoproliferation

The spleens of the mice were harvested and weighed after eight weeks of treatment. Splenomegaly of mice was evaluated by a spleen index (SI) (SI = (spleen weight/body weight) × 1000). The spleens were mashed and passed through a nylon cell strainer (BD Falcon, USA) to prepare a single-cell suspension. The cells were suspended in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum and centrifuged: the pelleted cells were resuspended in Lysing Buffer (BD Biosciences, USA) to lyse erythrocytes. After being washed in Phosphate Buffer Solution (PBS) the total number of cells was calculated and used for subsequent experiments.

Flow cytometry

Isolated spleen-cell suspensions were stained with fluorochrome-conjugated monoclonal antibodies against surface/intracellular markers: B cells (B220), T cells (CD90, CD3e, CD4, CD8, FoxP3), monocytes (CD11b), dentritic cells (CD11c), and appropriate isotypic antibodies (purchased from BD PharMingen, San Diego, California, USA) were used as controls. Activated CD4 + T and B cells were defined as CD69 + cells. For detection of the subsets of spleen cells, lymphocyte gates or spleen cell gates were established on the basis of linear FS (forward scatter) and SS (side scatter) to mark out the area in which the majority of such cells resided. A CD4 gate was set to mark out the area in which the majority of CD4 + FoxP3 + cells resided.

The percentage and the absolute number of positive stained cells were analyzed on a FACSCalibur flow cytometer using CellQuest pro software (Becton Dickinson, San Diego, California, USA).

Anti-DNA autoantibodies

Serum samples were collected every four weeks from 28-week-old BXSB mice. Serum anti-dsDNA IgG was measured with commercially available ELISA kits (ADI, San Antonio, USA) according to the manufacturer's instructions.

Cytokines detection

Interleukin-6 (IL-6), IL-10, IFN-γ, TNF-α, IL-12p70 and monocyte chemoattractant protein-1 (MCP-1) was detected using a mouse inflammation cytometric bead array kit (CBA; BD Biosciences, San Diego, California, USA) and was analyzed on a FACSCalibur flow cytometer. Standard curves were determined for each cytokine from a range of 10–5000 pg/ml. The lower limit of detection for the CBA, according to the manufacturer is 2.5–52.7 pg/ml, depended on the analyte.

Statistical analysis

Results were expressed as mean ± standard error (SE). Data analysis was performed using GraphPad Prism 5 version 5.01 software. The T test was used to compare the values. The value of p < 0.05 was considered as statistically significant.

Results

Effect of As₄S₄ treatment on clinical course and manifestations in BXSB mice

Skin lesions manifested as hair loss, erythema and scales on the face and ears which started at the age of 16 weeks. Histological examination revealed hyperkeratosis, epidermal hyperplasia and mononuclear cell infiltration of the dermis (Figure 1(A)). The skin lesions improved in BXSB mice treated with 1000 µg/g As₄S₄ and this was confirmed by histopathologic examination (Figure 1(B)). In the control group, three (3/10) mice died at 29 and 30 weeks: one of the treatment group mice died at 30 weeks. One (1/10) mouse developed arthritis with severe joint swelling, and this remitted after two weeks of As₄S₄ treatment (Figure 1(C) and (D)).

Effects of As₄S₄ treatment on histopathology of liver, lung and kidney in BXSB mice

Tissues of liver, lung and kidney were collected at the end of eight-week administration of As₄S₄ _. Histological examination exhibited hepatocyte spotty necrosis with lymphocyte and neutrophil infiltration around the portal and hepatic veins, which improved after eight weeks of treatment (Figure 2(A) and (B)). In C57BL/6 mice treated with 2000 µg/g As₄S₄, normal histological manifestation of livers was observed. As₄S₄ treatment slightly reduced the inflammation of lung disease in BXSB mice without significant difference compared to the control group.

Male BXSB mice develop severe lupus nephritis at 24 weeks of age.⁷ At 36 weeks of age, kidney histological examination exhibited mesangial proliferation, focal segmental glomerulosclerosis and tubular protein casts (Figure 3(A)). As₄S₄-treatment improved the change compared to the control group (Figure 3(B)). Strong deposition of IgG, IgM and C3 to the mesangium and capillary loop of glomeruli in BXSB mice was significantly inhibited with As₄S₄ treatment. As₄S₄-treatment decreased urinary albumin after four weeks of treatment, which was significant at week 7 (p > 0.05, Figure 3(C)).

Arsenic level in blood and hair

Blood and hair arsenic concentration was measured at day 1, 7, 20 and 60 of As₄S₄ administration. Arsenic was detectable in blood and hair after 24 h of treatment, and increased to 300.27 ng/ml in blood and 36.07 µg/g in hair on day 20 respectively (Figure 4(A) and (B)).

Effect of As₄S₄ on splenomegaly and spleen cell subsets

Splenomegaly was observed in adult BXSB mice. Treatment with As₄S₄ decreased the splenomegaly (Figure 5(A)). The weights of the spleens were 202.2 ± 14.71 mg in As₄S₄-treated BXSB mice and 223.22 ± 17.40 mg in control mice (p > 0.05). The body weights were 25.28 ± 0.54 g and 22.01 ± 0.86 g respectively (p > 0.05). The spleen index (SI) was 7.97 ± 0.55 and 10.32 ± 0.97 in As₄S₄-treated BXSB mice and the control group, respectively (p = 0.0447) (Figure 5(B)). As₄S₄ treatment showed improvement of splenomegaly, but not a return to normal (p < 0.05).

To detect toxicity of As₄S₄, spleen cell numbers and blood cell count were determined in BXSB and C57BL/6 mice treated with As₄S₄ for eight weeks. The total numbers of spleen cells in As₄S₄-treated BXSB mice were decreased, but slightly increased in C57BL/6 (p > 0.05) (Table 1). WBC were significantly decreased (p = 0.0328), and no change was observed in C57BL/6 mice.

Table 1

Tetra-arsenic tetra-sulfide (As₄S₄) effects on spleen cell count and peripheral cell number in BXSB and C57BL/6 mice

Spleen cell count (×10⁷)	WBC (×10⁹)	RBC (×10¹²)	PLT (×10⁹)
Control-BXSB	8.140 ± 0.485	14.602 ± 2.72	6.5 ± 0.297	564.6 ± 21.598
As₄S₄-treated BXSB	6.792 ± 0.757	7.7333 ± 1.067^a	5.635 ± 0.131	548.333 ± 33.734
Control-C57BL/6	5.538 ± 0.440	13.26 ± 1.3556	7.944 ± 0.214	418.4 ± 11.860
As₄S₄-treated C57BL/6	7.300 ± 1.206	13.54 ± 1.054	7.576 ± 0.391	497.6 ± 34.080

p < 0.05 compared with control BXSB mice (n ≥ 5). WBC: white blood cells; RBC: red blood cells; PLT: platelets

The spleen cell subsets of BXSB mice were measured using flow cytometric analysis. CD11b + cells were reduced both in percentage and absolute number in the As₄S₄-treated group (p = 0.0339, p = 0.0308 respectively). However, there was no significant difference in CD3 + T cells, CD4 + T cells, CD4 + FoxP3 + T cells, CD8 + T cells, B220 + B cells and CD11c + cells between the As₄S₄-treated and control groups. The active CD4 + T cells and B cells were slightly lower in As₄S₄-treated mice (p > 0.05) (Table 2).

Table 2

Phenotypes of spleen cells

Control-BXSB % Absolute number (×10⁵)		As₄S₄-treated-BXSB % Absolute number (×10⁵)
CD3	45.03 ± 1.96	205.96 ± 28.37	37.72 ± 3.02	188.55 ± 16.74
CD4	27.05 ± 1.70	122.33 ± 16.63	23.02 ± 1.90	115.81 ± 10.62
CD4CD69	0.66 ± 0.16	0.74 ± 0.22	0.59 ± 0.09	0.73 ± 0.139
CD4FOXP3	2.77 ± 0.28	3.44 ± 0.68	2.74 ± 0.38	3.23 ± 0.48
CD8	16.07 ± 1.57	74.48 ± 11.76	13.10 ± 2.06	64.92 ± 9.72
B220	36.98 ± 3.28	165.92 ± 21.57	35.00 ± 3.50	186.17 ± 25.36
B220CD69	1.67 ± 0.42	3.08 ± 1.28	1.56 ± 0.17	3.02 ± 0.57
CD11b	39.66 ± 6.97	333.20 ± 71.40	23.80 ± 1.54^a	166.50 ± 16.77^a
CD11c	4.13 ± 0.50	33.29 ± 3.97	3.71 ± 0.32	25.88 ± 3.15

p < 0.05 compared with control BXSB mice (n ≥ 5).

Effect of As₄S₄ treatment on serum anti-dsDNA antibodies and cytokines

Blood samples were collected at 28, 32 and 36 weeks. As₄S₄-treatment decreased the level of anti-dsDNA antibody slightly at the end of treatment, but without a significant difference (Figure (6)).

Serum levels of IL-6, IL-10, MCP-1, IFN-γ, TNF-α and IL-12p70 were analyzed in the BXSB and C57BL/6 mice. In BXSB mice, the IL-6 level was highly elevated (p = 0.0229). After treatment with As₄S₄, IL-6 reduced significantly (p = 0.0277, Figure (7)). The levels of IL-10, MCP-1, IFN-γ, TNF-α and IL-12p70 in As₄S₄ treatment groups were slightly lower than in the control group but the difference was not significant.

Discussion

As₄S₄, the major ingredient of realgar, has long been used in Chinese medicine for treatment of various diseases.⁸ In 1994, Lu et al.⁵ administered an oral preparation of highly purified As₄S₄ for the treatment of APL, and found support that it is effective on both remission induction and maintenance therapy for all stages of aPL with mild side effects. These findings give As₄S₄ an advantage over ATO in maintenance treatment. Previous studies reported that ATO was an effective agent for autoimmune disease treatment.^9,10 However, it is uncertain whether As₄S₄ had the same therapeutic effect as in those studies.

In our preliminary animal study, As₄S₄ showed efficacy with a dose of 1000 µg/g. In the present study, the effects of As₄S₄ on histology change, lymphocyte subsets, serum autoantibody and cytokines levels in BXSB mice were determined. The results suggested that oral As₄S₄ with the daily dose of 1000 µg/g could alleviate splenomegaly, skin lesions, liver lesions and nephritis. The monocytosis in BXSB mice was inhibited with a decreased level of IL-6. The treatment was tolerated well with mild adverse effects.

As₄S₄ is absorbed in minor amounts into the serum, where it mainly binds to sulfhydryl protein and other small molecules such as glutathione and cysteine, and is transported to various tissues and organs. It also binds to keratin and can be removed through hair, nail growth and cutaneous surfaces.¹¹ In this study, As₄S₄ concentration in plasma and hair increased after 24 h of treatment, and matched the treatment process, which suggested that the administration was effective.

Monocytosis might play an important role in the disease development of BXSB mice.^12–16 Our data revealed severe monocytosis developed (approximately 40% in the spleen), and decreased significantly after As₄S₄ treatment. The result suggests that arsenic might play a pivotal role in cell apoptosis induction and autophagy, through which monocytes were the major target.^17–19

As₄S₄ treatment improved glomerulonephritis in the BXSB kidney, confirmed by the decreased intensity of IgG, C3 and IgM deposition and the lower albumin in urine. However, the outcomes were not matched by the decrease of serum levels of anti-dsDNA antibodies. The results support the paradigm that anti-dsDNA antibody plays a major role in lupus nephritis and does not correlate with renal pathology in lupus-prone mice.²⁰

The liver is rarely involved in SLE patients (about 3–5%).^21,22 Andrew et al.³ reported that only one out of 20 BXSB mice developed cirrhosis at 12 ∼ 20 weeks old. A recent study indicated that Fas (TNF receptor superfamily member 6) and mitochondria dependent apoptosis increased in the livers of NZB/W F1 mice.²³ In our study, most BXSB mice developed hepatic histology at 36 weeks old. As₄S₄ reduced the liver inflammation of BXSB mice. It is known that ATO can cause severe liver damage, so we evaluated the liver toxicity of As₄S₄ in C57BL/6 mice. The result showed no obvious hepatotoxicity of As₄S₄ at the dose of 2000 µg/g. This is consistent with the result of mild and transient hepatotoxicity of As₄S₄ treatment in patients with aPL.⁵

Extensive cytokine dysregulation was demonstrated in BXSB mice.²⁴ In our study, serum levels of IL-6, IL-10, IL-12p70, IFN-γ, and TNF-α were detected. High levels of IL-6 were determined. IL-6 is a pleiotropic cytokine synthesized predominantly by macrophages and monocytes and involved in human lupus nephritis.^25,26 The significant decrease of IL-6 after As₄S₄ treatment was associated with the reduction of infiltrating macrophages in the kidney and decrease in renal IgG and C3 deposition.²⁷ These results indicate that IL-6 is a key factor in glomerulonephritis in lupus. As₄S₄ treatment contributed to this therapeutic effect.

BXSB mice are considered to be B cell-dominant in the development of autoimmune diseases.²⁸ No significant effect of As₄S₄ on B cells was observed in BXSB mice in our study. The outcomes suggest that monocytes might be involved in the pathogenesis of lupus in BXSB mice. These results are consistent with a prior report on monocytosis as strongly associated with autoantibody production and subsequent development of lupus nephritis.¹⁵ However, As₄S₄ did not affect lung disease in BXSB mice. The more likely possibility is that pathomechanism of individual end organs may not be as uniform, which confirms previous reports that had found dissociated outcomes for lung disease and other disease manifestations in lupus-prone mice.^29,30

This pilot study demonstrated the efficacy of As₄S₄ on autoimmune disease in BXSB mice. Clinically, As₄S₄ inhibited skin, kidney and liver involvements. Experimental study revealed that it decreased monocytosis and IL-6 production. No obvious side effect or toxicity was observed. The mechanism may overlap with, but not be identical to, that involved in the treatment of malignancies such as aPL, and needs further study to elucidate.

Footnotes

Funding

This study was supported by the Natural Science Foundation of China (No. 81071296) and the Beijing Natural Science Foundation (No. 7102156).

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Acknowledgements

The authors wish to express their gratitude to Professor Daopei Lu for his generous offer of As₄S₄.

References

Mills

. Systemic lupus erythematosus. N Engl J Med 1994; 330: 1871–1879.

Perry

Sang

Yin

. Murine models of systemic lupus erythematosus. J Biomed Biotechnol 2011; 2011: 1–19.

Andrews

Eisenberg

Theofilopoulos

. Spontaneous murine lupus-like syndromes. Clinical and immunopathological manifestations in several strains. J Exp Med 1978; 148: 1198–1215.

Karim

Pisoni

Khamashta

. Update on immunotherapy for systemic lupus erythematosus–what's hot and what's not!. Rheumatology (Oxford) 2009; 48: 332–341.

Qiu

Jiang

. Tetra-arsenic tetra-sulfide for the treatment of acute promyelocytic leukemia: A pilot report. Blood 2002; 99: 3136–3143.

Chen

. Comparative proteome study of apoptosis induced by As₄S₄ in retinoid acid resistant human acute promyelocytic leukemia NB4-R1 cells. Leuk Res 2010; 34: 1506–1516.

Theofilopoulos

Dixon

. Etiopathogenesis of murine SLE. Immunol Rev 1981; 55: 179–216.

Liu

. Mineral arsenicals in traditional medicines: Orpiment, realgar, and arsenolite. J Pharmacol Exp Ther 2008; 326: 363–368.

Bobe

Bonardelle

Benihoud

. Arsenic trioxide: A promising novel therapeutic agent for lymphoproliferative and autoimmune syndromes in MRL/lpr mice. Blood 2006; 108: 3967–3975.

10.

Xia

Lin

Zhou

. Effects of arsenic trioxide on the autoimmunity and survival time in BXSB lupus mice. Zhongguo Zhong Xi Yi Jie He Za Zhi 2007; 27: 138–141.

11.

Lindgren

Vahter

Dencker

. Autoradiographic studies on the distribution of arsenic in mice and hamsters administered 74As-arsenite or -arsenate. Acta Pharmacol Toxicol (Copenh) 1982; 51: 253–265.

12.

Wofsy

Kerger

Seaman

. Monocytosis in the BXSB model for systemic lupus erythematosus. J Exp Med 1984; 159: 629–634.

13.

Rogers

Gabriel

Nunes

. Monocytosis in BXSB mice is due to epistasis between Yaa and the telomeric region of chromosome 1 but does not drive the disease process. Genes Immun 2007; 8: 619–627.

14.

Merino

Fossati

Lacour

. H-2-linked control of the Yaa gene-induced acceleration of lupus-like autoimmune disease in BXSB mice. Eur J Immunol 1992; 22: 295–299.

15.

Kikuchi

Santiago-Raber

Amano

. Contribution of NZB autoimmunity 2 to Y-linked autoimmune acceleration-induced monocytosis in association with murine systemic lupus. J Immunol 2006; 176: 3240–3247.

16.

Granholm

Cavallo

. Bacterial lipopolysaccharide enhances deposition of immune complexes and exacerbates nephritis in BXSB lupus-prone mice. Clin Exp Immunol 1991; 85: 270–277.

17.

Lemarie

Morzadec

Bourdonnay

Fardel

Vernhet

. Human macrophages constitute targets for immunotoxic inorganic arsenic. J Immunol 2006; 177: 3019–3027.

18.

Goussetis

Altman

Glaser

. Autophagy is a critical mechanism for the induction of the antileukemic effects of arsenic trioxide. J Biol Chem 2010; 285: 29989–29997.

19.

Ondrousková

Soucek

Horváth

. Alternative pathways of programmed cell death are activated in cells with defective caspase-dependent apoptosis. Leuk Res 2008; 32: 599–609.

20.

Jacob

Zang

. Pivotal role of Stat4 and Stat6 in the pathogenesis of the lupus-like disease in the New Zealand mixed 2328 mice. J Immunol 2003; 171: 1564–1571.

21.

Chowdhary

Crowson

Poterucha

. Liver involvement in systemic lupus erythematosus: Case review of 40 patients. J Rheumatol 2008; 35: 2159–2164.

22.

Pistiner

Wallace

Nessim

. Lupus erythematosus in the 1980s: A survey of 570 patients. Semin Arthritis Rheum 1991; 21: 55–64.

23.

Tzang

Chiang

Lai

. Treatment with cystamine reduces apoptosis in liver from NZB/W F1 mice. Int Immunopharmacol 2008; 8: 589–596.

24.

Yap

Lai

. Cytokines and their roles in the pathogenesis of systemic lupus erythematosus: From basics to recent advances. J Biomed Biotechnol 2010; 2010: 1–10.

25.

Hirano

. Interleukin 6 and its receptor: Ten years later. Int Rev Immunol 1998; 16: 249–284.

26.

Takemura

Yoshioka

Murakami

. Cellular localization of inflammatory cytokines in human glomerulonephritis. Virchows Arch 1994; 424: 459–464.

27.

Cash

Relle

Menke

. Interleukin 6 (IL-6) deficiency delays lupus nephritis in MRL-Faslpr mice: The IL-6 pathway as a new therapeutic target in treatment of autoimmune kidney disease in systemic lupus erythematosus. J Rheumatol 2010; 37: 60–70.

28.

Smith

Chused

Smathers

. Evidence for thymic regulation of autoimmunity in BXSB mice: acceleration of disease by neonatal thymectomy. J Immunol 1983; 130: 1200–1204.

29.

Kulkarni

Ryu

Kantner

. Recombinant chaperonin 10 suppresses cutaneous lupus and lupus nephritis in MRL-(Fas)lpr mice. Nephrol Dial Transplant 2012; 27: 1358–1367.

30.

Kim

Ussin

Cheng

. TNFalpha inhibition in MRL/lpr mice ameliorates pulmonary but not renal disease. J Autoimmun 2002; 19: 215–222.