Lymphocyte sensitivity assay as a marker for glucocorticoid resistance in lupus: report of two sisters with systemic lupus erythematosus

Abstract

Glucocorticoid sensitivity can be measured in vitro using the lymphocyte sensitivity assay (LSA). In this test, dexamethasone is used to inhibit the proliferation of peripheral blood mononuclear cells (PBMC) in response to mitogens. If the proliferation of PBMC is suppressed the subjects are considered to be GC sensitive; if not, they are considered to be resistant. The LSA has been used to test GC sensitivity in some inflammatory diseases but its clinical value in systemic lupus erythematosus (SLE) has not been determined. Herein, we present the results of the LSA from two sisters with SLE who had different disease outcomes. Patient 1 presented with higher disease activity and damage accrual, and poorer response to corticosteroids than patient 2. In the LSA, patient 1 had a lower dexamethasone suppression of mitogen-stimulated PBMC than patient 2 and one control subject. The LSA could be helpful in identifying patients with GC resistance, thus allowing the consideration of alternative immunosuppressive drugs.

Keywords

Systemic lupus erythematosus clinical manifestations lymphocyte sensitivity assay glucocorticoid resistance

Introduction

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder characterized by autoreactive B cells and deregulation of T cells.¹ Patients with SLE have a wide spectrum of clinical manifestations and variable treatment response. Glucocorticoids (GCs) are the cornerstone in the treatment of SLE. They act by binding to the glucocorticoid receptor (GR), inhibiting the production of pro-inflammatory cytokines.² About 30% of patients with autoimmune conditions, such as SLE, have an impaired response to GCs.³ Therefore, screening for GC sensitivity early in the course of SLE might be helpful to identify those that are resistant to GC and that would require alternative immunosuppressive agents.

GC sensitivity can be measured in vitro using the lymphocyte sensitivity assay (LSA). In this test, dexamethasone is used to inhibit the proliferation of peripheral blood mononuclear cells (PBMC) in response to mitogens such as phytohemagglutinin or concavalin-A. If the proliferation of PBMC is suppressed the subjects are considered to be GC sensitive; conversely, if the proliferation is not inhibited, they are considered to be GC resistant.^4,5 The LSA has been used to test GC sensitivity in some autoimmune diseases but its clinical value in SLE has not been determined. Herein, we present the results of the LSA from two sisters with SLE who presented with different clinical outcomes and treatment response.

Case report

Patient 1

A 27-year-old woman was diagnosed with SLE at age 6 when she presented with malar rash, arthritis, leukopenia, lymphopenia, thrombocytopenia, and positive antinuclear (ANA), anti-Smith and anti-ribonucleoprotein antibodies. She was initially treated with hydroxychloroquine and prednisone 35 mg daily. Prednisone dose was gradually tapered down and discontinued six months later. Hydroxychloroquine was also discontinued. She remained stable until age 18 when she developed malar rash, photosensitivity, and discoid lesions. Two years later she developed nephrotic syndrome with proteinuria ranging between 10 and 15 g/day. Kidney biopsy showed membranous glomerulonephritis. She also developed antiphospholipid syndrome manifested by pulmonary embolism, elevated anticardiolipin antibodies, and positive lupus anticoagulant test that required chronic anticoagulation with warfarin. She was treated with high-dose prednisone (1 mg/kg/day) and intravenous pulse cyclophosphamide (0.75 g/m²/month) but this was discontinued after three months because of severe Herpes Zoster infection. She was continued on mid- to high-dose prednisone (30–60 mg daily) and hydroxychloroquine. Four years later she developed renal insufficiency. Prednisone was increased to 60 mg daily and she was started on azathioprine. She did not respond well to this treatment and azathioprine was switched to mycophenolate mofetil, which was continued for four years. Nonetheless, the patient progressed to end-stage renal disease requiring hemodialysis. Prednisone dose was gradually decreased to 20 mg daily; further reduction of this dose was not possible because of recurrence of nonrenal lupus manifestations.

Patient 2

A 21-year-old woman developed hematuria, proteinuria, and positive ANA at age 3. Kidney biopsy showed mesangial glomerulonephritis. She was treated with mid-dose prednisone (0.5 mg/kg/day) that was gradually reduced and discontinued one year later. She achieved complete remission and remained stable until age 18 when she developed photosensitivity, discoid lesions, oral ulcers, and seizures. Brain magnetic resonance imaging and angiography were consistent with vasculitis. She was treated with high-dose prednisone (1 mg/kg/day) and intravenous pulse cyclophosphamide (0.75 g/m²/month) for six months followed by maintenance therapy with azathioprine. Hydroxychloroquine was also added. Prednisone dose was gradually decreased to ≤10 mg daily. She remained stable having only mild exacerbations controlled with mid-dose prednisone.

LSA

Blood samples for the LSA were obtained from both SLE patients and one healthy woman. This study was approved by the institutional review board of the University of Puerto Rico Medical Sciences Campus. Table 1 shows the demographic parameters, clinical manifestations, disease activity, damage accrual, and pharmacologic treatments of the SLE patients at study visit. Patient 1 had higher disease activity and damage accrual than patient 2. Both patients were receiving a similar dose of prednisone, 20 and 15 mg daily, respectively.

Table 1

Demographic parameters, clinical manifestations, disease activity, damage accrual, and pharmacologic treatments of sisters with SLE

Feature	SLE patient 1	SLE patient 2
Age (years)	27	21
Gender	Female	Female
Disease duration (years)	21	18
Clinical manifestation
Mucocutaneous	Malar rash Discoid rash Photosensitivity Oral ulcers	Malar rash Discoid rash Photosensitivity Oral ulcers
Musculoskeletal	Arthritis	Arthralgias
Serositis	None	None
Renal	Diffuse membranous glomerulonephritis Nephrotic syndrome End-stage renal disease	Mesangial glomerulonephritis Microscopic hematuria Normal renal function
Neurologic	None	Seizures
Hematologic	Leukopenia Lymphopenia Thrombocytopenia Antiphospholipid syndrome (pulmonary embolism)	None
Vascular	Raynaud’s phenomenon	None
Features at study visit
Active clinical manifestations	Proteinuria 2 g/day	Discoid rash Arthralgias
Serologies	Positive ANA, anti-Sm, anti-RNP, aCL (IgG, IgM, IgA) antibodies Positive LA Normal C₃ and C₄ levels	Positive anti-beta2-GPI IgG Normal C₃ and C₄
SLAM	10	4
SDI	3	2
Pharmacologic treatment
Current immunosuppressive treatment	HCQ 400 mg/d MMF 1 g/d PRD 20 mg/d	HCQ 400 mg/d AZA 100 mg/d PRD 15 mg/d
Previous immunosuppressive treatment	Intravenous CYC AZA 50 mg/d	Intravenous CYC

SLE: systemic lupus erythematosus; ANA: antinuclear antibodies; anti-Sm: anti-Smith antibodies; anti-RNP: anti-ribonucleoprotein antibodies; aCL: anticardiolipin antibodies; LA: lupus anticoagulant test; antibeta2-GPI: anti-beta2-glycoprotein antibodies; WESR: Westergren sed rate; Ig: immunoglobulin; SLAM: Systemic Lupus Activity Measure; SDI: Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index; HCQ: hydroxychloroquine; AZA: azathioprine; MMF: mycophenolate mofetil; PRD: prednisone; CYC: cyclophosphamide.

The LSA was conducted as described before.^5,6 Briefly, 10 ml of peripheral blood were drawn from each subject. Using density centrifugation, PBMC were collected, then washed and suspended in Roswell Park Memorial Institute (RPMI) medium supplemented with penicillin/streptomycin, glutamine, HEPES buffer, and 5% fetal bovine serum (Sigma, St. Louis, MO). PBMC (2 × 10⁵ cells/well) were incubated in the presence or absence of 1 µg/ml phytohemagglutinin (PHA) (Sigma, St. Louis, MO), along with or without dexamethasone (Sigma, St. Louis, MO) at concentrations in the range of 10^–6 to 10⁻¹⁰mol/l. Assays were run in triplicates at 37℃ in a humidified atmosphere with 5% CO₂ for 72 hours. Twenty-four hours prior to the end of the incubation period, 1 µCi/ml of [³H]-thymidine (Moravek Biochemicals, Brea, CA) was added to the PBMC. Cells were harvested and 24 hours later scintillation fluid (EconoLume, Fair Lawn, NJ) was added. β-emission was measured in a scintillation counter and mean counts per minute (cpm) was calculated for each culture. Results were expressed in percentage of proliferation suppression [(mean cpm of PHA-stimulated PBMC in the absence of dexamethasone–mean cpm of PHA-stimulated PBMC in the presence of dexamethasone/mean cpm of PHA-stimulated PBMC in the absence of dexamethasone) ×100].

Figure 1 shows the effect of dexamethasone on PHA-stimulated PBMC derived from subjects. As expected, the dexamethasone suppression of PBMC proliferation was high for the healthy individual ranging from 71.4% to 95.2%. Patient 2 had a suppression similar to the control subject (range, 66.5–85.6). Conversely, patient 1 had a considerably lower suppression of PBMC proliferation in all concentrations of dexamethasone (range, 36.9%–55.7%) when compared to patient 2 and the control subject.

Discussion

GC resistance is a phenomenon observed with frequency in SLE and is associated with increased disease activity and damage.⁷ In the present report we used the LSA to determine the GC sensitivity in two sisters with SLE. Patient 1 presented with higher disease activity and damage accrual and poorer response to corticosteroid treatment than patient 2. In the LSA, patient 1 had a lower dexamethasone suppression of mitogen-stimulated PBMC than patient 2 and one control subject. At study visit, both patients were receiving a similar dose of GCs.

GCs work by several mechanisms, including blocking transcription factors of several cytokines such as interleukin (IL)-1, IL-2, IL-3, IL-4, tumor necrosis factor (TNF) α, interferon (IFN) γ and granulocyte colony-stimulating factor, and inducing apoptosis of T lymphocytes.⁸ The mechanisms of GC resistance are numerous, including mutations and polymorphisms of the GR gene,^9–11 defective GR binding and translocation resulting in altered transcription and expression of pro-inflammatory genes,^12,13 and polymorphisms in genes involved in the transport and/or metabolism of GCs.¹⁴

Different methods have been used to assess GC resistance in inflammatory conditions such as asthma, ulcerative colitis, and rheumatoid arthritis.^4,12,15 These tests range from screening assays such as the LSA to tests that explore specific resistance mechanisms. In ulcerative colitis the LSA helps to predict treatment failure.⁶ Also, in patients with rheumatoid arthritis the LSA has shown to be a useful tool in predicting the therapeutic effect of GCs.^4,5

Some tests similar to the LSA have been studied in SLE to determine the PBMC and cytokine profile in GC-resistant patients. However, these studies were not intended to examine the clinical value of the LSA. For example, isolated PBMC were cultured at different concentrations of GCs after being stimulated with anti-CD3 monoclonal antibody. Lymphocytes that survived in assays from steroid-resistant patients were found to be mostly CD8+ and Bcl-2+.⁸ In a study to evaluate the expression of the GR in T lymphocytes and monocytes of SLE patients, the inhibitory effect of dexamethasone on PHA-stimulated PBMC was determined to assess GR sensitivity.¹⁶ GR expression and binding were decreased in cells from SLE patients that were steroid resistant. In vitro analysis of cytokines showed suppression of TNF-α, IL-12 and INF-γ with dexamethasone in patients classified as controls and steroid sensitive compared to GC-resistant patients.

Because the mechanisms of GC resistance are complex, concurrent studies to corroborate the value of the LSA would be necessary. For example, the LSA will be more informative if associated with GC sensitivity determined by measuring the effects of GCs on transactivation of the GC-induced leucine zipper gene and on transrepression of the IL-2 gene.¹⁷ Unfortunately, other tests determining GC sensitivity or resistance were not performed on our patients.

In summary, our case report suggests that the LSA is a reliable tool to predict the responsiveness to GC therapy in SLE patients. SLE patients who have GC resistance could have a more severe disease than those who are GC sensitive. Of concern is the fact that although the anti-inflammatory properties of GCs are diminished in GC-resistant patients, these patients are still susceptible to deleterious adverse events of these drugs.¹⁸ The LSA could be helpful in identifying patients with GC resistance, thus allowing the consideration of alternative immunosuppressive drugs, and at the same time, preventing or reducing the long-term adverse events of GCs. Nonetheless, these observations must be confirmed with clinical studies in large SLE cohorts.

Footnotes

Funding

This work was supported by an unrestricted educational grant from Bristol-Myers Squibb Puerto Rico Inc.

Conflict of interest

The authors have no conflicts of interest to declare.

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