International Journal of Rheumatic Diseases 2014; 17: 788–793
ORIGINAL ARTICLE
Increase of peripheral T regulatory cells during remission induction with cyclophosphamide in active systemic lupus erythematosus Tselios KONSTANTINOS,1 Sarantopoulos ALEXANDROS,1 Gkougkourelas IOANNIS,1 Papagianni AIKATERINI2 and Boura PANAGIOTA1 1
Clinical Immunology Unit, 2nd Department of Internal Medicine, Aristotle University of Thessaloniki, and 2Department of Nephrology, Hippokration General Hospital, Aristotle University of Thessaloniki, Thessaloniki, Greece
Abstract Background: Cyclophosphamide efficacy in lupus nephritis (LN) and neuropsychiatric systemic lupus erythematosus (NPSLE) is probably mediated by a non-specific ablation of reactive lymphocytes. However, little is known in regard to its effect on T regulatory cells (Tregs) in such patients, which was the aim of this study. Patients and Methods: Ten Caucasian lupus patients were included, six with LN classes IV–V (mean age 33.8 8.8 years) and four with NPSLE (mean age 35.5 8.8 years, clinical manifestations: 1/4 acute confusional state, 1/4 psychosis, 2/4 refractory seizures). Cyclophosphamide was administered at monthly pulses (500 mg/m2/month for 6 months); doses of other administered drugs, including steroids, remained stable or lower. CD4+CD25highFOXP3+ Tregs were assessed by flow-cytometry at baseline and before every subsequent pulse and 3–6 months after the final pulse. Disease activity was assessed by SLE Disease Activity Index (SLEDAI). Results: In LN patients, Tregs were significantly increased even after the fourth pulse (0.54 0.20% vs. 1.24 0.29%, P < 0.001). Likewise, in NPSLE, Tregs were significantly expanded after the fourth pulse (0.57 0.23% vs. 1.41 0.28%, P < 0.001). SLEDAI was significantly reduced in all patients. Conclusions: Cyclophosphamide pulse therapy was associated with a significant increase of the CD4+CD25highFOXP3+ Tregs in patients with active LN and NPSLE. This effect is probably indirect and may partially explain the beneficial role of cyclophosphamide in such cases. Key words: systemic lupus erythematosus, T regulatory cells, cyclophosphamide.
INTRODUCTION Systemic lupus erythematosus (SLE) represents a prototype systemic autoimmune disease with various clinical manifestations and a wide variety of immunological abnormalities. Among clinical features, lupus nephritis (LN) and neuropsychiatric involvement (neuropsychiCorrespondence: Konstantinos Tselios, MD, PhD, Aristotle University of Thessaloniki, 2nd Department of Internal Medicine, Hippokration General Hospital, Konstantinoupoleos St. 49, 546 42 Thessaloniki, Greece. Email: [email protected]
atric SLE, NPSLE) are considered to decisively affect prognosis.1 Cyclophosphamide, usually in combination with steroids, has been proven to be efficacious in remission induction in such cases, with a response rate of almost 80%.2,3 In LN particularly, cyclophosphamide is believed to be the standard of care in patients with progressive disease.2,4 Current recommendations support the administration of low doses (6 9 500 mg/m2 per 2 weeks, Euro-Lupus Nephritis Trial protocol) which have comparable efficacy with a higher dose every 21 days, regimen (6 9 750 1000 mg/m2 National Institutes of Health protocol, USA).5
© 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
Cyclophosphamide and Tregs in SLE
Given that SLE represents the final result of the breakdown of immune tolerance, which is mainly maintained by peripheral T regulatory cells (Tregs), several studies have reported impaired numbers and function of these cells in lupus patients.6–8 In addition, these cells were found to be inversely correlated to disease activity, while their numbers were restored after successful treatment.6,9 However, little is known about the effects of certain drugs in this cellular population, other than steroids.9,10 In the present study, the effect of cyclophosphamide pulse therapy on CD4+CD25highFOXP3+ Tregs was prospectively investigated in patients with active LN and NPSLE.
PATIENTS AND METHODS Ten White Caucasian patients (nine females, one male) were included in the study, six with active LN and four with active NPSLE. All patients fulfilled the updated American College of Rheumatology diagnostic criteria for SLE.11 Remission induction therapy consisted of intravenous cyclophosphamide (pulse therapy, 500 mg/ m2/month for 6 months); the doses of steroids and other drugs remained stable or lower throughout the study period. Lupus nephritis patients (five female, one male, mean age 33.8 8.8 years, mean disease duration 87.2 63.4 months) were suffering from classes IV–V nephritis, according to the International Society of Nephrology/Renal Pthology Society (ISN/RPS) classification.12 In parallel with cyclophosphamide, patients were administered methylprednisolone (32 mg/day) with slow tapering. After remission induction, maintenance therapy included steroids (methylprednisolone 8–32 mg/day in all patients), azathioprine (100 mg/day in four patients) and mycophenolate mofetil (1080– 1440 mg/day in two patients). Neuropsychiatric systemic lupus erythematosus patients (four females, mean age 35.5 8.8 years, mean disease duration 103.8 55.1 months) were administered cyclophosphamide pulse therapy because of acute confusional state (one patient), refractory seizures (two patients) and psychosis (one patient). Standard oral therapy included methylprednisolone (16–64 mg/day, with slow tapering in parallel with cyclophosphamide) and azathioprine (100 mg/day, stable dose). Anti-convulsants were used concomitantly in the two patients with refractory seizures and anti-psychotic drugs in the other two patients. The Human Ethics Review Committee of Aristotle University of Thessaloniki approved the study protocol
International Journal of Rheumatic Diseases 2014; 17: 788–793
and signed informed consent was obtained from each subject.
Laboratory methods and study design At baseline and before every subsequent cyclophosphamide administration, Tregs (phenotype CD4+CD25highFOXP3+) were assessed by triple-color flow cytometry (Fluorescence Activated Cell Sorter, FACS, EPICS COULTER XLâ, Leriva Diagnostics, Athens, Greece), in whole blood samples, shortly after venepuncture. Cells were stained with anti-CD4 (13B8.2, Immunotech, Leriva Diagnostics, Athens, Greece) FITC (fluoroscein isothiocyanate), anti-CD25 (B1.49.9, Immunotech), ECD (Phycoerythrin-Texas-Red-X) and anti-FOXP3 (PCH101, e-Bioscience, Varelas SA, Thessaloniki, Greece) PE (phycoerythrin). Intraprep TM solutions (Beckman-Coulter, Leriva Diagnostics, Athens, Greece) were used to increase leukocyte membrane permeability and induce red blood cell lysis. A full blood count with leukocyte differential was performed before each measurement in order to quantify CD4+ T cells and Tregs (given as a proportion of the CD4+ T cells and as absolute numbers). Additional laboratory parameters included full blood count, erythrocyte sedimentation rate (ESR), serum creatinine and proteinuria (24-h urine collection, only in LN patients), C3/C4d ***complement components (nephelometry, Dade Behring, Newark, DE, USA) and anti-double-stranded DNA (anti-dsDNA) antibodies (immunofluorescence assay, IFA, on a Crithidia luciliae substrate). Clinical assessment was made using the SLE Disease Activity Index (SLEDAI). All the aforementioned parameters were assessed monthly for the first five pulses and 3–6 months after the last cyclophosphamide pulse.
Statistical analysis Analysis was performed using the non-parametric Kruskal–Wallis test for independent variables and Monte Carlo simulation. All P-values were two-tailed and P < 0.05 was considered to be statistically significant. The SPSS software package (version 20.0; SPSS Inc., Chicago, IL, USA) was used.
RESULTS Cyclophosphamide leads to significant Tregs expansion in active LN A gradual increase in CD4+CD25highFOXP3+ Tregs numbers was observed from pulse to pulse (Fig. 1). Tregs, as a proportion of CD4+ T cells, were significantly
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as assessed by serum creatinine, remained normal throughout the study. Proteinuria was significantly decreased (baseline 2576 3985 mg/24 h vs. after the sixth pulse 234 86 mg/24 h, P < 0.05). Contrary to Tregs, SLEDAI was substantially decreased (baseline 15.2 6.8 vs. after the sixth pulse 2 1.8, P < 0.05). The exact variations of Tregs, SLEDAI and levels of proteinuria from pulse to pulse are shown in Table 1.
Cyclophosphamide leads to significant Tregs expansion in active NPSLE
Figure 1 Individual pulse-to-pulse variation of T regulatory cells (Tregs) in lupus nephritis (LN) patients.
increased even from the fifth cyclophosphamide pulse (baseline 0.54 0.20% compared to 1.24 0.29% after the fourth pulse, P < 0.05). Absolute Tregs counts were also increased (3 1.4 cells/mm3 to 10.7 4.6 cells/mm3 after the sixth pulse, P < 0.05). White blood cells (WBC) were increased (4697 1637/mm3 to 7460 1853/mm3 after the sixth pulse, P < 0.05), along with absolute lymphocyte count (965 438/mm3 to 1657 999/mm3 after the sixth pulse, P = 0.083). Erythrocyte sedimentation rate was marginally decreased (baseline 43.5 19.2 mm/h compared to 23.8 11.3 mm/h after the sixth pulse, P = 0.052). Concerning C3 and C4d levels, these were insignificantly increased between baseline and the end of therapy (C3 95.4 65.3 mg/dL vs. 153.5 43.1 mg/dL, P = 0.155, C4d 9 8.15 mg/dL vs. 18.5 9.2 mg/dL, P = 0.117). Regarding anti-dsDNA autoantibodies, these were positive in 4/6 patients; in all of them these remained positive after the sixth pulse. Renal function,
CD4+CD25highFOXP3+ Tregs were substantially increased from pulse to pulse (Table 1, Fig. 2), while they were significantly increased even from the fifth cyclophosphamide pulse (baseline 0.57 0.23% compared to 1.41 0.28% after the fourth pulse, P < 0.05). Absolute Tregs counts were also increased
Figure 2 Individual pulse-to-pulse variation of T regulatory cells (Tregs) in neuropsychiatric systemic lupus erythematosus (NPSLE) patients.
Table 1 Cyclophosphamide pulse-to-pulse variations of Tregs, proteinuria and SLEDAI Tregs LN Baseline 1st pulse 2nd pulse 3rd pulse 4th pulse P 5th pulse P After 6th pulse P
0.54 0.70 0.88 0.96 0.04 1.24 < 0.001 1.23 < 0.001
0.20 0.20 0.33 0.31 0.29 0.29
SLEDAI LN
Proteinuria
Tregs NPSLE
SLEDAI NPSLE
15.2 10.3 8 8.4 0.033 2.8 < 0.001 2 < 0.001
2576 2600 1008 809 0.008 366 0.006 234 0.002
0.57 0.62 1.11 1.14 0.014 1.41 < 0.001 1.39 < 0.001
17.8 16.3 6.5 4 0.008 2.3 < 0.001 2.1 < 0.001
6.8 2.7 3.7 2.2 1.8 1.6
3985 3058 565 267 85 86
0.23 0.10 0.46 0.31 0.28 0.25
7.7 7.5 1.9 2.8 1.7 1.6
LN, lupus nephritis; NPSLE, neuropsychiatric systemic lupus erythematosus; SLEDAI, Systemic Lupus Erythematosus Disease Activity Index. Units: Tregs (%CD4+ T cells), proteinuria (mg/24 h).
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(4 2.2 cells/mm3 to 9 2.3 cells/mm3 after the sixth pulse, P < 0.05). WBCs were increased (4640 1573/ mm3 to 6700 3083/mm3 after the sixth pulse, P = 0.14), whereas absolute lymphocyte count to remained unchanged (1141 650/mm3 3 1115 330/mm after the sixth pulse). Erythrocyte sedimentation rate was gradually decreased during therapy (baseline 58.5 15.6 mm/h vs. 27.5 20.5 mm/h after the sixth pulse, P = 0.026). In addition, C3 and C4d levels were increased between baseline and the end of therapy (C3 103.8 33 mg/dL vs. 130 36 mg/dL, P = 0.181, C4d 13.5 8.7 mg/dL vs. 20 8.9 mg/dL, P = 0.188). In contrast, SLEDAI was substantially decreased (baseline 17.8 7.7 vs. after the sixth pulse 2.1 1.6, P < 0.05). The exact variations of Tregs and SLEDAI from pulse to pulse are shown in Table 1.
DISCUSSION Cyclophosphamide is an alkylating agent, which inhibits DNA replication and, subsequently, cell division. Its effect is considered to be more prominent in highly proliferating cells, such as stimulated lymphocytes.13 However, little is known in regard to its impact on specific lymphocyte subpopulations, such as Tregs. Cyclophosphamide has been extensively used in systemic autoimmune diseases with visceral involvement, in particular, vasculitis and SLE.14 Pulse therapy is considered to exert similar efficacy in oral regimens, albeit with a favorable safety profile.5 In the present study, we investigated the effect of cyclophosphamide on CD4+CD25highFOXP3+ Tregs in active lupus patients. We observed that these cells were significantly increased after four pulses of cyclophosphamide in LN patients, while this increment was accompanied by a significant decrease in disease activity and proteinuria. In LN, most studies demonstrated significantly lower numbers of Tregs in active disease,15–18 while others failed to confirm these results.19 In one study, Treg depletion was accompanied by a compensatory expansion of Th17 cells and related cytokines interleukin (IL)-17 and IL-23.17 In accordance with these results, relevant research demonstrated a reduction in transforming growth factor (TGF)-b levels in active LN, a cytokine known to be essential for Tregs functional differentiation.18 In regard to the influence of certain immunosuppressive drugs on Tregs in LN, early studies in lupus-prone mice reported no alteration or even decreased T regulatory cells (phenotype CD4+CD25+CD45RBlow) during cyclophosphamide treatment.20 In the only study
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in lupus patients, CD4+CD25high regulatory T cells were increased after treatment with cyclophosphamide and corticosteroids.21 However, the clinical phenotype of active lupus patients, for example, LN or NPSLE, was not specified in this study. Given that corticosteroids are able to drive an expansion of Tregs in SLE,9,10 it can be assumed that this was reasonably the cause of Tregs increment in that study.21 Concerning other drugs currently used in LN, rituximab led to an increase of Tregs, which accompanied clinical remission.15,16 Concerning NPSLE, the other major indication for cyclophosphamide administration,22 there is no study in human lupus to investigate the effect of cyclophosphamide on Tregs. In the present study, cyclophosphamide administration resulted in a significant Tregs increment after the fourth pulse, whereas this expansion was followed by clinical remission in all patients. Again, in the study by Zhang et al., a significant increase of Tregs in lupus patients was reported after cyclophosphamide treatment, without specifying the exact organ involved.21 Despite the lack of evidence in regard to cyclophosphamide’s effect on Tregs in systemic autoimmune diseases, contradictory results were reported from studies in cancer. In this case, cyclophosphamide exerts a more complicated effect on immune cells than previously considered.23 Beside its tumoricidal effect in high doses, this agent demonstrated a potent immunosuppressive function via lymphocyte ablation. In cancer patients, previous studies reported Tregs depletion, along with reduction of FOXP3 expression, after cyclophosphamide therapy.24,25 However, this effect is decisively dependent on the drug dose, as it was demonstrated that low doses (250 mg/m2) led to Tregs decrement, while higher doses (500 and 750 mg/m2) failed to reduce these cells in patients with metastatic carcinoma.26 Similar findings were drawn from a study in hepatocellular carcinoma, where intermediate doses (350 mg/m2) failed to drive the apoptosis of Tregs.27 The discrepancy between the results of the present study and previous reports in neoplastic diseases may be explained by the individual characteristics of patients with SLE and cancer. In active SLE, low levels of Tregs have been reported by most studies.6–9 In opposition, in cancer, increased numbers of these cells have been described in the peripheral blood of patients with hematopoietic and solid tumors.28–30 In this case, Tregs are able to infiltrate the tumor via a chemokine (CCL22) gradient and the expression of CCR4 on their surface.31 In addition, it is possible that a significant portion of these cells is induced by CD4+CD25 na€ıve T cells in the periphery.32 Thus, it can be hypothesized
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that these inducible Tregs may be more sensitive to cyclophosphamide-induced Fas-mediated apoptosis, as in the case of proliferating lymphocytes. In the present study, Tregs were assessed by using the CD4+CD25highFOXP3+ phenotype, which is believed to better characterize thymic-derived, natural Tregs than inducible Tregs.33 These natural Tregs display an anergic state with a low proliferative potential, thus conferring resistance to cyclophosphamide. Another important issue is the lack of evidence for the kinetics of Tregs after cyclophosphamide administration. It has been reported that, in mice, Tregs depletion lasted for 45 days after cyclophosphamide administration.34 In patients with hepatocellular carcinoma, in which low doses of cyclophosphamide (250 mg/m2) were administered, the time to Tregs restoration was estimated to be 8 and 21 days.27 In this context, it could be assumed that Tregs may slightly decline in the next few days after cyclophosphamide administration, and then are restored and even increase in 1 month, as was observed in the present study. Another confounding factor may be the concomitant corticosteroids that were administered in our patients. Corticosteroids have been shown to significantly increase Tregs in lupus patients.9,10 In addition, steroids were used concomitantly with cyclophosphamide in the only published study addressing the effect of this drug on Tregs in lupus patients.21 In this regard, Tregs increment may be attributed to the combined result of methylprednisolone and cyclophosphamide, as steroid doses remained stable or lower throughout the study. Finally, given that cyclophosphamide leads to nonspecific lymphocyte ablation, it could be hypothesized that its net effect on lymphocyte subpopulations depends on the relative effect on Tregs and their effector counterparts, for instance Th1 and Th17 cells. In SLE, it has been demonstrated that the dynamic balance between Th17 and Tregs is of paramount importance in disease pathogenesis.35 Furthermore, the relative expression of certain cytokines, such as IL-6 and TGF-b, may drive the differentiation of Th17 cells (in case of IL-6 predominance) or Tregs (in case of TGF-b predominance). In this regard, it seems reasonable that cyclophosphamide may lead to Tregs expansion by suppressing Th17 cells and enhancing TGF-b production, a hypothesis proven in multiple sclerosis, an organ-specific autoimmune disease.36 Based on these data, it seems justifiable that Tregs expansion, after cyclophosphamide pulse therapy, represents an indirect effect of this agent. Limitations to be considered in the present study include the small number of patients, which does not
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allow for definite conclusions, and the lack of functional assays for better characterization of Tregs. Concomitant steroid use also raises questions about the exact effect of cyclophosphamide on Tregs, as discussed above. In conclusion, cyclophosphamide pulse therapy leads, over time, to a significant increase in CD4+CD25highFOXP3+ T regulatory cells in active lupus patients with progressive nephritis or neuropsychiatric involvement. This expansion is probably secondary to SLE remission; however, it may partially explain the beneficial role of this drug in such cases. Future studies are warranted to elucidate the molecular basis of these findings and promote our understanding of the immunomodulative effects of cyclophosphamide.
CONFLICTS OF INTEREST STATEMENT The authors declare there is no conflict of interest. This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
REFERENCES 1 Tsokos GC (2011) Systemic lupus erythematosus. N Engl J Med 365, 2110–21. 2 Houssiau FA (2012) Therapy of lupus nephritis: lessons learned from clinical research and daily care of patients. Arthritis Res Ther 14, 202. 3 Popescu A, Kao AH (2011) Neuropsychiatric systemic lupus erythematosus. Curr Neuropharmacol 9, 449–57. 4 Petri M, Brodsky RA, Jones RJ, Gladstone D, Fillius M, Magder LS (2010) High-dose cyclophosphamide versus monthly intravenous cyclophosphamide for systemic lupus erythematosus: a prospective randomized trial. Arthritis Rheum 62, 487–93. 5 Bertsias GK, Tektonidou M, Amoura Z et al. (2012) Joint European League Against Rheumatism and European Renal Association-European Dialysis and Transplant Association (EULAR/ERA-EDTA) recommendations for the management of adult and paediatric lupus nephritis. Ann Rheum Dis 71, 1771–82. 6 Miyara M, Amoura Z, Parizot C et al. (2005) Global natural regulatory T cell depletion in active systemic lupus erythematosus. J Immunol 175, 8392–400. 7 Lyssouk EY, Torgashina AV, Soloviev SK, Nassonov EL, Bykovskaia SN (2007) Reduced number and function of CD4 + CD25highFOXP3 + regulatory T cells in patients with systemic lupus erythematosus. Adv Exp Med Biol 601, 113–9. 8 Valencia X, Yarboro C, Illei G, Lipsky PE (2007) Deficient CD4 + CD25high T regulatory cell function in patients with active systemic lupus erythematosus. J Immunol 178, 2579–88.
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9 Tselios K, Sarantopoulos A, Gkougkourelas I, Boura P (2014) CD4 + CD25highFOXP3 + T regulatory cells as a biomarker of disease activity in systemic lupus erythematosus: a prospective study. Clin Exp Rheumatol 2014 Sep 8 [Epub ahead of print] PMID: 25197969. 10 Prado C, Gomez J, Lopez P, De Paz B, Gutierrez C, Suarez A (2011) Dexamethasone upregulates FOXP3 expression without increasing regulatory activity. Immunobiology 216, 386–92. 11 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 40, 1725. 12 Weening JJ, D’Agati VD, Schwartz MM et al. (2004) The classification of glomerulonephritis in systemic lupus erythematosus revisited. Kidney Int 65, 521–30. 13 Pinto N, Ludeman SM, Dolan ME (2009) Drug focus: pharmacogenetic studies related to cyclophosphamidebased therapy. Pharmacogenomics 10, 1897–903. 14 Jayne D (2012) Treating vasculitis with conventional immunosuppressive agents. Cleve Clin J Med 79(Suppl.3), S46. 15 Vignas-Perez M, Hernandez-Castro B, Paredes-Saharopoulos O et al. (2006) Clinical and immunological effects of rituximab in patients with lupus nephritis refractory to conventional therapy: a pilot study. Arthritis Res Ther 8, R83. 16 Sfikakis PP, Souliotis VL, Fragiadaki KG, Moutsopoulos HM, Boletis JN, Theofilopoulos AN (2007) Increased expression of the FoxP3 functional marker of regulatory T cells following B cell depletion with rituximab in patients with lupus nephritis. Clin Immunol 123, 66–73. 17 Xing Q, Wang B, Su H, Cui J, Li J (2012) Elevated Th17 cells are accompanied by FoxP3 + Treg cells decrease in patients with lupus nephritis. Rheumatol Int 32, 949–58. 18 Edelbauer M, Kshirsagar S, Riedl M et al. (2012) Acivity of childhood lupus nephritis is linked to altered T cell and cytokine homeostasis. J Clin Immunol 32, 477–87. 19 Yates J, Whittington A, Mitchell P, Lechler RI, Lightstone L, Lombardi G (2008) Natural regulatory T cells: number and function are normal in the majority of patients with lupus nephritis. Clin Exp Immunol 153, 44–55. 20 Sharabi A, Azulai H, Sthoeger ZM, Mozes E (2007) Clinical amelioration of murine lupus by a peptide based on the complementarity determining region-1 of an autoantibody and by cyclophosphamide: similarities and differences in the mechanisms of action. Immunology 121, 248–57. 21 Zhang B, Zhang X, Tang F, Zhu L, Liu Y (2008) Reduction of forkhead box P3 levels in CD4 + CD25high T cells in patients with new-onset systemic lupus erythematosus. Clin Exp Immunol 153, 182–7. 22 Bertsias G, Ioannidis JPA, Boletis J et al. (2008) EULAR recommendations for the management of systemic lupus erythematosus. Report of a Task Force of the EULAR Standing Committee for International Clinical Studies including Therapeutics. Ann Rheum Dis 67, 195–205.
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23 Sharabi A, Haran-Ghera N (2011) Immune recovery after cyclophosphamide treatment in multiple myeloma: implication for maintenance immunotherapy. Bone Marrow Res 2011, 269519. 24 Ghiringelli F, Lamonier N, Schmitt E et al. (2004) CD4 + CD25 + regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative. Eur J Immunol 34, 336–44. 25 Lutsiak MEC, Semanni RT, De Pascalis R, Kashmiri SVS, Schlom J, Sabzevari H (2005) Inhibition of CD4 + CD25 + T regulatory cell function implicated in enhanced immune response by low-dose cyclophosphamide. Blood 105, 2862–8. 26 Audia S, Nicolas A, Cathelin D et al. (2007) Increase of CD4 + CD25 + regulatory T cells in the peripheral blood of patients with metastatic carcinoma: a phase I clinical trial using cyclophosphamide and immunotherapy to eliminate CD4 + CD25 + T lymphocytes. Clin Exp Immunol 150, 523–30. 27 Greten TF, Ormandy LA, Fikuart A et al. (2010) Lowdose cyclophosphamide treatment impairs regulatory T cells and unmasks AFP-specific CD4 + T-cell responses in patients with advanced HCC. J Immunother 33, 211–8. 28 Wu CP, Qing X, Wu CY, Zhu H, Zhou HY (2012) Immunophenotype and increased presence of CD4(+)CD25(+) regulatory T cells in patients with acute lymphoblastic leukaemia. Oncol Lett 3, 421–4. 29 Cheng HH, Tseng GY, Yang HB, Wang HJ, Lin HJ, Wang WC (2012) Increased numbers of Foxp3-positive regulatory T cells in gastritis, peptic ulcer and gastric adenocarcinoma. World J Gastroenterol 18, 34–43. 30 Thakur S, Singla A, Chawla Y, Rajwanshi A, Kalra N, Arora SK (2011) Expansion of peripheral and intratumoral regulatory T cells in hepatocellular carcinoma: a case-control study. Indian J Pathol Microbiol 54, 448–53. 31 Peng DJ, Liu R, Zou W (2012) Regulatory T cells in ovarian cancer. J Oncol 2012, 345164. 32 Han Y, Wu J, Bi L et al. (2011) Malignant B cells induce the conversion of CD4 + CD25- T cells to regulatory T cells in B-cell non-Hodgkin lymphoma. PLoS One 6, e28649. 33 Lourenco EV, La Cava A (2011) Natural regulatory T cells in autoimmunity. Autoimmunity 44, 33–42. 34 Sharabi A, Laronne-Bar-On A, Meshorer A, Haran-Ghera N (2010) Chemoimmunotherapy reduces the progression of multiple myeloma in a mouse model. Cancer Prev Res 3, 1265–76. 35 Alunno A, Bartoloni E, Bistoni O et al. (2012) Balance between regulatory T and Th17 cells in systemic lupus erythematosus: the old and the new. Clin Dev Immunol 2012, 823085. 36 Elkhalifa A, Weiner H (2010) Cyclophosphamide treatment of MS: current therapeutic approaches and treatment regimens. Int MS J 17, 12–8.
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ORIGINAL ARTICLE
Increase of peripheral T regulatory cells during remission induction with cyclophosphamide in active systemic lupus erythematosus Tselios KONSTANTINOS,1 Sarantopoulos ALEXANDROS,1 Gkougkourelas IOANNIS,1 Papagianni AIKATERINI2 and Boura PANAGIOTA1 1
Clinical Immunology Unit, 2nd Department of Internal Medicine, Aristotle University of Thessaloniki, and 2Department of Nephrology, Hippokration General Hospital, Aristotle University of Thessaloniki, Thessaloniki, Greece
Abstract Background: Cyclophosphamide efficacy in lupus nephritis (LN) and neuropsychiatric systemic lupus erythematosus (NPSLE) is probably mediated by a non-specific ablation of reactive lymphocytes. However, little is known in regard to its effect on T regulatory cells (Tregs) in such patients, which was the aim of this study. Patients and Methods: Ten Caucasian lupus patients were included, six with LN classes IV–V (mean age 33.8 8.8 years) and four with NPSLE (mean age 35.5 8.8 years, clinical manifestations: 1/4 acute confusional state, 1/4 psychosis, 2/4 refractory seizures). Cyclophosphamide was administered at monthly pulses (500 mg/m2/month for 6 months); doses of other administered drugs, including steroids, remained stable or lower. CD4+CD25highFOXP3+ Tregs were assessed by flow-cytometry at baseline and before every subsequent pulse and 3–6 months after the final pulse. Disease activity was assessed by SLE Disease Activity Index (SLEDAI). Results: In LN patients, Tregs were significantly increased even after the fourth pulse (0.54 0.20% vs. 1.24 0.29%, P < 0.001). Likewise, in NPSLE, Tregs were significantly expanded after the fourth pulse (0.57 0.23% vs. 1.41 0.28%, P < 0.001). SLEDAI was significantly reduced in all patients. Conclusions: Cyclophosphamide pulse therapy was associated with a significant increase of the CD4+CD25highFOXP3+ Tregs in patients with active LN and NPSLE. This effect is probably indirect and may partially explain the beneficial role of cyclophosphamide in such cases. Key words: systemic lupus erythematosus, T regulatory cells, cyclophosphamide.
INTRODUCTION Systemic lupus erythematosus (SLE) represents a prototype systemic autoimmune disease with various clinical manifestations and a wide variety of immunological abnormalities. Among clinical features, lupus nephritis (LN) and neuropsychiatric involvement (neuropsychiCorrespondence: Konstantinos Tselios, MD, PhD, Aristotle University of Thessaloniki, 2nd Department of Internal Medicine, Hippokration General Hospital, Konstantinoupoleos St. 49, 546 42 Thessaloniki, Greece. Email: [email protected]
atric SLE, NPSLE) are considered to decisively affect prognosis.1 Cyclophosphamide, usually in combination with steroids, has been proven to be efficacious in remission induction in such cases, with a response rate of almost 80%.2,3 In LN particularly, cyclophosphamide is believed to be the standard of care in patients with progressive disease.2,4 Current recommendations support the administration of low doses (6 9 500 mg/m2 per 2 weeks, Euro-Lupus Nephritis Trial protocol) which have comparable efficacy with a higher dose every 21 days, regimen (6 9 750 1000 mg/m2 National Institutes of Health protocol, USA).5
© 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
Cyclophosphamide and Tregs in SLE
Given that SLE represents the final result of the breakdown of immune tolerance, which is mainly maintained by peripheral T regulatory cells (Tregs), several studies have reported impaired numbers and function of these cells in lupus patients.6–8 In addition, these cells were found to be inversely correlated to disease activity, while their numbers were restored after successful treatment.6,9 However, little is known about the effects of certain drugs in this cellular population, other than steroids.9,10 In the present study, the effect of cyclophosphamide pulse therapy on CD4+CD25highFOXP3+ Tregs was prospectively investigated in patients with active LN and NPSLE.
PATIENTS AND METHODS Ten White Caucasian patients (nine females, one male) were included in the study, six with active LN and four with active NPSLE. All patients fulfilled the updated American College of Rheumatology diagnostic criteria for SLE.11 Remission induction therapy consisted of intravenous cyclophosphamide (pulse therapy, 500 mg/ m2/month for 6 months); the doses of steroids and other drugs remained stable or lower throughout the study period. Lupus nephritis patients (five female, one male, mean age 33.8 8.8 years, mean disease duration 87.2 63.4 months) were suffering from classes IV–V nephritis, according to the International Society of Nephrology/Renal Pthology Society (ISN/RPS) classification.12 In parallel with cyclophosphamide, patients were administered methylprednisolone (32 mg/day) with slow tapering. After remission induction, maintenance therapy included steroids (methylprednisolone 8–32 mg/day in all patients), azathioprine (100 mg/day in four patients) and mycophenolate mofetil (1080– 1440 mg/day in two patients). Neuropsychiatric systemic lupus erythematosus patients (four females, mean age 35.5 8.8 years, mean disease duration 103.8 55.1 months) were administered cyclophosphamide pulse therapy because of acute confusional state (one patient), refractory seizures (two patients) and psychosis (one patient). Standard oral therapy included methylprednisolone (16–64 mg/day, with slow tapering in parallel with cyclophosphamide) and azathioprine (100 mg/day, stable dose). Anti-convulsants were used concomitantly in the two patients with refractory seizures and anti-psychotic drugs in the other two patients. The Human Ethics Review Committee of Aristotle University of Thessaloniki approved the study protocol
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and signed informed consent was obtained from each subject.
Laboratory methods and study design At baseline and before every subsequent cyclophosphamide administration, Tregs (phenotype CD4+CD25highFOXP3+) were assessed by triple-color flow cytometry (Fluorescence Activated Cell Sorter, FACS, EPICS COULTER XLâ, Leriva Diagnostics, Athens, Greece), in whole blood samples, shortly after venepuncture. Cells were stained with anti-CD4 (13B8.2, Immunotech, Leriva Diagnostics, Athens, Greece) FITC (fluoroscein isothiocyanate), anti-CD25 (B1.49.9, Immunotech), ECD (Phycoerythrin-Texas-Red-X) and anti-FOXP3 (PCH101, e-Bioscience, Varelas SA, Thessaloniki, Greece) PE (phycoerythrin). Intraprep TM solutions (Beckman-Coulter, Leriva Diagnostics, Athens, Greece) were used to increase leukocyte membrane permeability and induce red blood cell lysis. A full blood count with leukocyte differential was performed before each measurement in order to quantify CD4+ T cells and Tregs (given as a proportion of the CD4+ T cells and as absolute numbers). Additional laboratory parameters included full blood count, erythrocyte sedimentation rate (ESR), serum creatinine and proteinuria (24-h urine collection, only in LN patients), C3/C4d ***complement components (nephelometry, Dade Behring, Newark, DE, USA) and anti-double-stranded DNA (anti-dsDNA) antibodies (immunofluorescence assay, IFA, on a Crithidia luciliae substrate). Clinical assessment was made using the SLE Disease Activity Index (SLEDAI). All the aforementioned parameters were assessed monthly for the first five pulses and 3–6 months after the last cyclophosphamide pulse.
Statistical analysis Analysis was performed using the non-parametric Kruskal–Wallis test for independent variables and Monte Carlo simulation. All P-values were two-tailed and P < 0.05 was considered to be statistically significant. The SPSS software package (version 20.0; SPSS Inc., Chicago, IL, USA) was used.
RESULTS Cyclophosphamide leads to significant Tregs expansion in active LN A gradual increase in CD4+CD25highFOXP3+ Tregs numbers was observed from pulse to pulse (Fig. 1). Tregs, as a proportion of CD4+ T cells, were significantly
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as assessed by serum creatinine, remained normal throughout the study. Proteinuria was significantly decreased (baseline 2576 3985 mg/24 h vs. after the sixth pulse 234 86 mg/24 h, P < 0.05). Contrary to Tregs, SLEDAI was substantially decreased (baseline 15.2 6.8 vs. after the sixth pulse 2 1.8, P < 0.05). The exact variations of Tregs, SLEDAI and levels of proteinuria from pulse to pulse are shown in Table 1.
Cyclophosphamide leads to significant Tregs expansion in active NPSLE
Figure 1 Individual pulse-to-pulse variation of T regulatory cells (Tregs) in lupus nephritis (LN) patients.
increased even from the fifth cyclophosphamide pulse (baseline 0.54 0.20% compared to 1.24 0.29% after the fourth pulse, P < 0.05). Absolute Tregs counts were also increased (3 1.4 cells/mm3 to 10.7 4.6 cells/mm3 after the sixth pulse, P < 0.05). White blood cells (WBC) were increased (4697 1637/mm3 to 7460 1853/mm3 after the sixth pulse, P < 0.05), along with absolute lymphocyte count (965 438/mm3 to 1657 999/mm3 after the sixth pulse, P = 0.083). Erythrocyte sedimentation rate was marginally decreased (baseline 43.5 19.2 mm/h compared to 23.8 11.3 mm/h after the sixth pulse, P = 0.052). Concerning C3 and C4d levels, these were insignificantly increased between baseline and the end of therapy (C3 95.4 65.3 mg/dL vs. 153.5 43.1 mg/dL, P = 0.155, C4d 9 8.15 mg/dL vs. 18.5 9.2 mg/dL, P = 0.117). Regarding anti-dsDNA autoantibodies, these were positive in 4/6 patients; in all of them these remained positive after the sixth pulse. Renal function,
CD4+CD25highFOXP3+ Tregs were substantially increased from pulse to pulse (Table 1, Fig. 2), while they were significantly increased even from the fifth cyclophosphamide pulse (baseline 0.57 0.23% compared to 1.41 0.28% after the fourth pulse, P < 0.05). Absolute Tregs counts were also increased
Figure 2 Individual pulse-to-pulse variation of T regulatory cells (Tregs) in neuropsychiatric systemic lupus erythematosus (NPSLE) patients.
Table 1 Cyclophosphamide pulse-to-pulse variations of Tregs, proteinuria and SLEDAI Tregs LN Baseline 1st pulse 2nd pulse 3rd pulse 4th pulse P 5th pulse P After 6th pulse P
0.54 0.70 0.88 0.96 0.04 1.24 < 0.001 1.23 < 0.001
0.20 0.20 0.33 0.31 0.29 0.29
SLEDAI LN
Proteinuria
Tregs NPSLE
SLEDAI NPSLE
15.2 10.3 8 8.4 0.033 2.8 < 0.001 2 < 0.001
2576 2600 1008 809 0.008 366 0.006 234 0.002
0.57 0.62 1.11 1.14 0.014 1.41 < 0.001 1.39 < 0.001
17.8 16.3 6.5 4 0.008 2.3 < 0.001 2.1 < 0.001
6.8 2.7 3.7 2.2 1.8 1.6
3985 3058 565 267 85 86
0.23 0.10 0.46 0.31 0.28 0.25
7.7 7.5 1.9 2.8 1.7 1.6
LN, lupus nephritis; NPSLE, neuropsychiatric systemic lupus erythematosus; SLEDAI, Systemic Lupus Erythematosus Disease Activity Index. Units: Tregs (%CD4+ T cells), proteinuria (mg/24 h).
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(4 2.2 cells/mm3 to 9 2.3 cells/mm3 after the sixth pulse, P < 0.05). WBCs were increased (4640 1573/ mm3 to 6700 3083/mm3 after the sixth pulse, P = 0.14), whereas absolute lymphocyte count to remained unchanged (1141 650/mm3 3 1115 330/mm after the sixth pulse). Erythrocyte sedimentation rate was gradually decreased during therapy (baseline 58.5 15.6 mm/h vs. 27.5 20.5 mm/h after the sixth pulse, P = 0.026). In addition, C3 and C4d levels were increased between baseline and the end of therapy (C3 103.8 33 mg/dL vs. 130 36 mg/dL, P = 0.181, C4d 13.5 8.7 mg/dL vs. 20 8.9 mg/dL, P = 0.188). In contrast, SLEDAI was substantially decreased (baseline 17.8 7.7 vs. after the sixth pulse 2.1 1.6, P < 0.05). The exact variations of Tregs and SLEDAI from pulse to pulse are shown in Table 1.
DISCUSSION Cyclophosphamide is an alkylating agent, which inhibits DNA replication and, subsequently, cell division. Its effect is considered to be more prominent in highly proliferating cells, such as stimulated lymphocytes.13 However, little is known in regard to its impact on specific lymphocyte subpopulations, such as Tregs. Cyclophosphamide has been extensively used in systemic autoimmune diseases with visceral involvement, in particular, vasculitis and SLE.14 Pulse therapy is considered to exert similar efficacy in oral regimens, albeit with a favorable safety profile.5 In the present study, we investigated the effect of cyclophosphamide on CD4+CD25highFOXP3+ Tregs in active lupus patients. We observed that these cells were significantly increased after four pulses of cyclophosphamide in LN patients, while this increment was accompanied by a significant decrease in disease activity and proteinuria. In LN, most studies demonstrated significantly lower numbers of Tregs in active disease,15–18 while others failed to confirm these results.19 In one study, Treg depletion was accompanied by a compensatory expansion of Th17 cells and related cytokines interleukin (IL)-17 and IL-23.17 In accordance with these results, relevant research demonstrated a reduction in transforming growth factor (TGF)-b levels in active LN, a cytokine known to be essential for Tregs functional differentiation.18 In regard to the influence of certain immunosuppressive drugs on Tregs in LN, early studies in lupus-prone mice reported no alteration or even decreased T regulatory cells (phenotype CD4+CD25+CD45RBlow) during cyclophosphamide treatment.20 In the only study
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in lupus patients, CD4+CD25high regulatory T cells were increased after treatment with cyclophosphamide and corticosteroids.21 However, the clinical phenotype of active lupus patients, for example, LN or NPSLE, was not specified in this study. Given that corticosteroids are able to drive an expansion of Tregs in SLE,9,10 it can be assumed that this was reasonably the cause of Tregs increment in that study.21 Concerning other drugs currently used in LN, rituximab led to an increase of Tregs, which accompanied clinical remission.15,16 Concerning NPSLE, the other major indication for cyclophosphamide administration,22 there is no study in human lupus to investigate the effect of cyclophosphamide on Tregs. In the present study, cyclophosphamide administration resulted in a significant Tregs increment after the fourth pulse, whereas this expansion was followed by clinical remission in all patients. Again, in the study by Zhang et al., a significant increase of Tregs in lupus patients was reported after cyclophosphamide treatment, without specifying the exact organ involved.21 Despite the lack of evidence in regard to cyclophosphamide’s effect on Tregs in systemic autoimmune diseases, contradictory results were reported from studies in cancer. In this case, cyclophosphamide exerts a more complicated effect on immune cells than previously considered.23 Beside its tumoricidal effect in high doses, this agent demonstrated a potent immunosuppressive function via lymphocyte ablation. In cancer patients, previous studies reported Tregs depletion, along with reduction of FOXP3 expression, after cyclophosphamide therapy.24,25 However, this effect is decisively dependent on the drug dose, as it was demonstrated that low doses (250 mg/m2) led to Tregs decrement, while higher doses (500 and 750 mg/m2) failed to reduce these cells in patients with metastatic carcinoma.26 Similar findings were drawn from a study in hepatocellular carcinoma, where intermediate doses (350 mg/m2) failed to drive the apoptosis of Tregs.27 The discrepancy between the results of the present study and previous reports in neoplastic diseases may be explained by the individual characteristics of patients with SLE and cancer. In active SLE, low levels of Tregs have been reported by most studies.6–9 In opposition, in cancer, increased numbers of these cells have been described in the peripheral blood of patients with hematopoietic and solid tumors.28–30 In this case, Tregs are able to infiltrate the tumor via a chemokine (CCL22) gradient and the expression of CCR4 on their surface.31 In addition, it is possible that a significant portion of these cells is induced by CD4+CD25 na€ıve T cells in the periphery.32 Thus, it can be hypothesized
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that these inducible Tregs may be more sensitive to cyclophosphamide-induced Fas-mediated apoptosis, as in the case of proliferating lymphocytes. In the present study, Tregs were assessed by using the CD4+CD25highFOXP3+ phenotype, which is believed to better characterize thymic-derived, natural Tregs than inducible Tregs.33 These natural Tregs display an anergic state with a low proliferative potential, thus conferring resistance to cyclophosphamide. Another important issue is the lack of evidence for the kinetics of Tregs after cyclophosphamide administration. It has been reported that, in mice, Tregs depletion lasted for 45 days after cyclophosphamide administration.34 In patients with hepatocellular carcinoma, in which low doses of cyclophosphamide (250 mg/m2) were administered, the time to Tregs restoration was estimated to be 8 and 21 days.27 In this context, it could be assumed that Tregs may slightly decline in the next few days after cyclophosphamide administration, and then are restored and even increase in 1 month, as was observed in the present study. Another confounding factor may be the concomitant corticosteroids that were administered in our patients. Corticosteroids have been shown to significantly increase Tregs in lupus patients.9,10 In addition, steroids were used concomitantly with cyclophosphamide in the only published study addressing the effect of this drug on Tregs in lupus patients.21 In this regard, Tregs increment may be attributed to the combined result of methylprednisolone and cyclophosphamide, as steroid doses remained stable or lower throughout the study. Finally, given that cyclophosphamide leads to nonspecific lymphocyte ablation, it could be hypothesized that its net effect on lymphocyte subpopulations depends on the relative effect on Tregs and their effector counterparts, for instance Th1 and Th17 cells. In SLE, it has been demonstrated that the dynamic balance between Th17 and Tregs is of paramount importance in disease pathogenesis.35 Furthermore, the relative expression of certain cytokines, such as IL-6 and TGF-b, may drive the differentiation of Th17 cells (in case of IL-6 predominance) or Tregs (in case of TGF-b predominance). In this regard, it seems reasonable that cyclophosphamide may lead to Tregs expansion by suppressing Th17 cells and enhancing TGF-b production, a hypothesis proven in multiple sclerosis, an organ-specific autoimmune disease.36 Based on these data, it seems justifiable that Tregs expansion, after cyclophosphamide pulse therapy, represents an indirect effect of this agent. Limitations to be considered in the present study include the small number of patients, which does not
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allow for definite conclusions, and the lack of functional assays for better characterization of Tregs. Concomitant steroid use also raises questions about the exact effect of cyclophosphamide on Tregs, as discussed above. In conclusion, cyclophosphamide pulse therapy leads, over time, to a significant increase in CD4+CD25highFOXP3+ T regulatory cells in active lupus patients with progressive nephritis or neuropsychiatric involvement. This expansion is probably secondary to SLE remission; however, it may partially explain the beneficial role of this drug in such cases. Future studies are warranted to elucidate the molecular basis of these findings and promote our understanding of the immunomodulative effects of cyclophosphamide.
CONFLICTS OF INTEREST STATEMENT The authors declare there is no conflict of interest. This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
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