Introduction
Lupus erythematosus (LE) is a chronic autoimmune inflammatory disease. Skin involvement occurs in 70% to 85% of all patients with LE [
1]. Cutaneous disease is classified as LE-specific or LE-nonspecific, based on assessments of the morphology of the cutaneous lesions and the results of histopathologic examinations [
2]. LE-specific skin lesions are divided into three broad categories that include chronic cutaneous lupus erythematosus (CCLE), subacute cutaneous lupus erythematosus (SCLE) and acute cutaneous lupus erythematosus [
3]. Herein we focus on two subsets of CCLE: (1) discoid lupus erythematosus (DLE) and tumid lupus erythematosus (TLE) and (2) SCLE. DLE typically presents as scaly, erythematous, disk-shaped patches and plaques, whereas TLE manifests as single or multiple raised polycyclic erythematous plaques with a bright reddish or violaceous smooth surface that does not scar [
1]. SCLE typically presents as erythematous papulosquamous, psoriasiform plaques or annular-polycyclic plaques and the lesions typically resolve without scarring [
4]. Dermatomyositis (DM) is a chronic inflammatory disorder of the skin and muscles, with histologic findings similar to SCLE [
5,
6].
TNFα is a critical proinflammatory cytokine that is implicated in the pathogenesis of multiple inflammatory diseases. TNFα can be produced by many different cell types including monocytes, macrophages, dendritic cells, T and B lymphocytes, natural killer cells, neutrophils, mast cells, endothelial cells, fibroblasts and keratinocytes [
7‐
9]. TNFα is produced as pro-TNFα, which is expressed on the plasma membrane, where it can be cleaved in the extracellular domain by ADAM17 (also known as "TACE," or TNFα-converting enzyme), which is a matrix metalloproteinase, and results in the release of TNFα in a soluble form [
10]. ADAM17 mRNA expression was found to be increased in lesional skin from psoriasis patients [
11]. Both membrane-associated and soluble TNFα are active [
5].
Several studies have examined TNFα in skin lesions and serum of patients with CLE and DM. TNFα is increased in lesional skin of patients with DLE and SCLE compared to controls [
12‐
15]. DM lesional skin expresses TNFα, but staining has been found to be more evident in DLE patients and absent in control specimens [
5]. TNFα was increased in muscle biopsies of DM patients [
16]. TNFα has been reported to be elevated in sera of some patients with systemic lupus erythematosus (SLE), DLE and SCLE, but not in that of patients with DM [
17‐
20].
In vitro production of TNFα by unstimulated PBMCs from juvenile DM patients was measured in a previous study in which the investigators found that PBMCs from children who had a disease course ≥ 36 months produced more TNFα compared to PBMCs from juvenile DM patients who had had the disease < 36 months [
21]. As there are data regarding TNFα expression in the skin and sera of CLE and DM patients, but, to the best of our knowledge, minimal data regarding TNFα production from the PBMCs of these patients, the aim of our study was to evaluate the production of TNFα by PBMCs from DLE, SCLE, TLE and DM patients.
Materials and methods
Subjects
Twenty-one DLE, ten SCLE, ten TLE and eighteen DM patients were recruited from the outpatient autoimmunity clinic in the Department of Dermatology at the Hospital of the University of Pennsylvania. All subjects who had the diagnosis of DLE, SCLE, TLE and/or DM were invited to participate in the study. Seven of the DLE patients and one of the SCLE patients had SLE as well. All of the recruited patients were receiving therapy to treat their disease (see Table
1). Eighteen people without LE, DM or any autoimmune disease were recruited as controls. To be eligible for the study, controls must not have been on corticosteroids or immunosuppressive drugs.
Table 1
Patient demographics and clinical characteristicsa
DLE (n = 21) | Females, 16 Males, 5 | AA, 12 White, 8 Hispanic, 1 | 45.0 (23 to 64) | 8 | Antimalarials, 18 Methotrexate, 2 Immunosuppressants, 5 Corticosteroids, 3 Dapsone, 1 Thalidomide, 1 |
SCLE (n = 10) | Females, 7 Males, 3 | White, 9 Asian, 1 | 47.1 (20 to 64) | 9.5 | Antimalarials, 7 Immunosuppressants, 2 Corticosteroids, 2 Thalidomide, 4 |
TLE (n = 10) | Females, 8 Males, 2 | AA, 1 White, 9 | 42.9 (25 to 57) | 10.1 | Antimalarials, 10 Methotrexate, 1 |
DM (n = 18) | Females, 15 Males, 3 | AA, 1 White, 16 Asian, 1 | 49 (21 to 75) | 14.7 | Antimalarials, 10 Methotrexate, 6 Immunosuppressants, 6 Corticosteroids, 9 |
controls (n = 18) | Females, 10 Males, 8 | AA, 4 White, 12 Asian, 2 | 31.2 (26 to 57) | N/A | N/A |
Demographic information and data regarding current medications and comorbid illnesses were collected from all subjects. The treating dermatologist evaluated each LE and DM patient by performing a complete physical examination and assessing the patient based on Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI) and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores for LE patients and the Cutaneous Dermatomyositis Area and Severity Index (CDASI) scores for DM patients to measure disease activity and damage [
22,
23]. This study was approved by the Institutional Review Board (IRB) at our institution, and all PBMCs were obtained according to the IRB protocol. The Declaration of Helsinki protocols were followed, and patients gave their written, informed consent.
Cell culture and TNFα determination by ELISA
PBMCs were separated from heparinized venous blood of patients with DLE, SCLE, TLE and DM, as well as from controls, on an endotoxin-free Ficoll-Paque PLUS™ gradient (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). PBMCs were cultured overnight in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% L-glutamine and 1% penicillin-streptomycin at 37°C and 5% CO2. The following morning PBMCs (1 × 105 cells in 0.2 ml per well) were plated in 96-well plates (Falcon, Franklin Lakes, NJ, USA) and cultured for 24 hours at 37°C and 5% CO2. Conditioned media were then collected and stored at -80°C. TNFα was quantitated by ELISA according to the manufacturer's protocol (BD Biosciences, San Diego, CA, USA). All determinations were performed in triplicate. The viability of cells was measured by trypan blue staining.
Flow cytometric analysis
Prior to intracellular staining for TNFα, PBMCs were cultured in medium for 4 hours in the presence of brefeldin A (1 μg/ml; Invitrogen/Life Technologies, Carlsbad, CA, USA). To detect intracellular expression of TNFα in dendritic cells, approximately 106 PBMCs per sample were stained with lineage 1 cocktail-fluorescein isothiocyanate (FITC) (lineage cocktail containing antibodies against CD3, CD14, CD16, CD19, CD20 and CD56), anti-human leukocyte antigen (HLA)-DR-perdinin-chlorophyll-protein (PerCp) and anti-CD123-phycoerythrin (PE) (plasmacytoid dendritic cells (pDCs)) or anti-CD11c-PE (myeloid dendritic cells (mDCs)) for 30 minutes on ice, followed by 15-minute incubation with fixation reagent A (FIX & PERM; Invitrogen/Life Technologies). Cells were then washed, permeabilized with reagent B (FIX & PERM; Invitrogen Life/Technologies) and stained with anti-TNFα-allophycocyanin (anti-TNFα-APC) antibody (or an appropriate isotype control) for 15 minutes at room temperature.
To detect intracellular expression of TNFα in monocytes, PBMCs were stained with anti-CD64-FITC and anti-CD14-PE followed by anti-TNFα-APC or an appropriate isotype control as described above. To detect intracellular expression of TNFα in T cells, PBMCs were stained with anti-CD3-FITC followed by anti-TNFα-APC or an appropriate isotype control as described above. All antibodies used were purchased from BD Biosciences.
Cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) with BD CellQuest software (BD Biosciences) at the Flow Cytometry and Cell Sorting Core, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA. We acquired 150,000 events of live cells.
Statistical analysis
One-way analysis of variance followed by Dunnett's multiple comparisons test was used for statistical evaluation as appropriate. P values less than 0.05 in the post hoc test were considered significant. Correlations were calculated by performing a Pearson correlation test.
Discussion
The proinflammatory nature of TNFα in inflammatory diseases is well-established (reviewed in [
7]). TNFα causes the upregulation of adhesion molecules and chemokines, which leads to attachment of inflammatory cells to vessels, rolling, emigration and eventually chemotaxis [
24‐
26]. TNFα is an activating cytokine for fibroblasts and leukocytes, including dendritic cells. TNFα stimulation leads to increased production of IFN-γ, a cytokine that is also increased in lesional skin of DLE patients [
12]. Our study demonstrates that TNFα release is significantly greater in PBMCs from DLE patients compared to controls.
We next determined which specific cell type was secreting the increased TNFα in DLE patients. Our flow cytometry results demonstrated that the majority of TNFα was produced by monocytes and mDCs, with a statistically significant difference between TNFα intracellular staining from mDCs of DLE patients (P < 0.001) and DM patients (P < 0.001) compared to controls. A significantly greater MFI from monocytes and mDCs from DLE patients compared to controls was also present, whereas there was no statistically significant difference in MFI from monocytes and mDCs between DM patients and controls. The significant elevation of the MFI in DLE patients compared to controls demonstrates that the monocytes and mDCs from DLE patients are synthesizing TNFα in greater quantities compared to controls. The MFI of the PBMCs from DLE patients is significantly greater than that of controls. Our ELISA results demonstrate an approximately 15-fold increase in TNFα release from DLE PBMCs compared to those seen in DM, and the flow cytometry results explain this finding in part. We realize the monocytes and mDCs comprise a relatively small percentage of circulating cells and that there may be another population of cells that we did not stain that could contribute to the increased TNFα release in DLE. We will investigate other cell types in the future.
We next explored whether there were any differences between the size of the monocyte and mDC populations among the DLE and DM patients and controls. We found a trend toward an increased population of monocytes in DLE patients compared to both DM patients and controls. The mDC population was trending toward greater numbers in DLE patients compared to controls. These flow cytometry results support our cultured PBMC ELISA results, which showed that there was a significantly greater amount of TNFα released from DLE patients. The increased amount of TNFα in monocytes and mDCs from DLE patients and the greater size of these cell populations in DLE patients may further explain why PBMCs from DLE patients release greater quantities of TNFα.
Plasmacytoid DCs have been implicated in the pathogenesis of both SLE and CLE. SLE is characterized by elevated levels of IFNα and IFNβ (both type I IFNs) [
27,
28]. Because pDCs are the main cell type producing IFNα and IFNβ, they have been implicated in disease pathogenesis. A higher proportion of pDCs are also present in the skin than in the blood of patients with SLE and CLE, suggesting that pDCs migrate from the circulation to the skin and may play a role in the production of skin lesions [
29,
30]. Our experiments demonstrate that TNFα levels produced by PBMCs vary with the different CLE subsets and with DM. We found that pDCs produced minimal amounts of TNFα compared to monocytes and mDCs. Therefore, we postulate that monocytes and mDCs may have a bigger role in the pathogenesis of DLE than was originally believed. The increased numbers of inflammatory cells seen in the skin of DLE patients relative to DM patients and controls may be explained, at least in part, by the increased TNFα produced in DLE, given the importance of TNFα in cell recruitment to endothelial cells and skin [
25].
Flow cytometry showed that both CD11c
+HLA-DR
hi and CD11c
+HLA-DR
low mDCs produced TNFα and accounted for much of the TNFα production exhibited by PBMCs. Recently, an inflammatory mDC known as Tip-DC (TNFα-inducible nitric oxide synthase-producing dendritic cells) has been found in elevated amounts in psoriasis plaques compared with nonlesional skin from psoriasis patients and normal skin [
9,
31]. These Tip-DCs are CD11c
+, BDCA-1
- and HLA-DR
+ [
31]. Tip-DCs express HLA-DR at 10-fold lower levels compared to CD11c
+ and BDCA-1
+ mDCs. It is possible that some of the CD11c
+HLA-DR
low mDCs in our DLE patients might actually have been Tip-DCs. It has been shown that bone marrow-derived monocytes accumulate and differentiate to Tip-DCs in the spleen, liver and lymph nodes of
Trypanosoma brucei brucei-infected mice [
32]. Another type of inflammatory mDCs, known as M-DC8
+ cells, has also recently been described that are CD11c
+, CD14
+, CD16
+, CD64
- and HLA-DR
+ [
33,
34]. These M-DC8
+ cells are able to produce significantly greater amounts of TNFα in response to LPS compared to M-DC8
- monocytes and DCs [
8,
35]. Although we have not directly addressed these two populations of mDCs in our current studies, there is a possibility that these populations might contribute to enhanced TNFα production in DLE patients. Further studies designed to determine a potential role of these predominantly tissue-based DCs are being performed.
Acknowledgements
This study was supported in part by a merit review grant from the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development, and by National Institutes of Health (NIH) grant K24-AR 02207 (to VPW).). In addition, funding was provided by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
ASN and MMB carried out the PBMC cultures and TNF ELISA studies. AB and MW participated in the flow studies, and AB drafted the manuscript. VPW performed the CLASI assessments. VPW and ASN participated in the design of the study. MS performed the statistical analysis. VPW conceived the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.