Tumor necrosis factor ? release in peripheral blood mononuclear cells of cutaneous lupus and dermatomyositis patients.

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by  Nabatian, Adam S

Nabatianetal.ArthritisResearch&Therapy2012,14:R1
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RESEARCH ARTICLE Open Access
Tumor necrosis factor a release in peripheral
blood mononuclear cells of cutaneous lupus and
dermatomyositis patients
Adam S Nabatian1,2,3, Muhammad M Bashir1,2, Maria Wysocka2, Meena Sharma1,2 and Victoria P Werth1,2*
Abstract
Introduction: Several studies have reported that TNFa is substantially increased within skin lesions of patients with
discoid lupus erythematosus (DLE), subacute cutaneous lupus erythematosus (SCLE) and dermatomyositis (DM)
compared to controls. Elevated TNFa has been reported in the sera of some patients with systemic lupus
erythematosus, DLE and SCLE, but not in the sera of patients with DM. Because of the key pathogenic role of
autoimmunity in these diseases, in this study we sought to evaluate TNFa production by a readily available source
of immune cells (namely, peripheral blood mononuclear cells (PBMCs)) taken from controls and from patients with
cutaneous lupus or DM.
Methods: Freshly isolated PBMCs were cultured overnight, and TNFa protein accumulation in conditioned
medium was determined. In addition, flow cytometry using cell-type-specific markers was performed to determine
the sources of TNFa. One-way analysis of variance and Dunnett’s multiple comparisons test were performed for
statistical comparisons.
Results: Accumulation of TNFa protein in conditioned medium containing PBMCs from DLE patients, but not from
SCLE, TLE or DM patients, was significantly greater (19-fold) than that from controls (P < 0.001). In DLE PBMCs,
increased TNFa was produced by circulating monocytes and myeloid dendritic cells (mDCs). The mean TNFa
fluorescence intensity, but not the total number, of both monocytes and mDCs (P < 0.01) from DLE patients was
significantly greater (2.3-fold) than that of controls. There were significantly more (13.3-fold) mDCs with intracellular
TNFa in blood from DLE patients (P < 0.001) and DM patients (P < 0.001) compared to controls. Most importantly,
a positive correlation was seen in DLE patients between their disease activity measured using the Cutaneous Lupus
Erythematosus Disease Area and Severity Index and TNFa protein secretion (r = 0.61, P < 0.08).
Conclusions: TNFa protein production by PBMCs is greater in DLE patients than in patients with other cutaneous
forms of lupus and DM or in controls. Flow cytometric studies demonstrated that circulating monocytes and mDCs
contributed to this increased TNFa production. Monocytes and mDCs are present in lesional skin, and the
increased TNFa production by these cells and other PBMCs likely increase the number of inflammatory cells seen
in DLE skin relative to other subsets of cutaneous lupus erythematosus and DM. These results provide a possible
biological explanation for the denser infiltrate seen in DLE relative to DM.
Introduction assessments of the morphology of the cutaneous lesions
Lupus erythematosus (LE) is a chronic autoimmune and the results of histopathologic examinations [2]. LE-
inflammatory disease. Skin involvement occurs in 70% specific skin lesions are divided into three broad cate-
to 85% of all patients with LE [1]. Cutaneous disease is gories that include chronic cutaneous lupus erythemato-
classified as LE-specific or LE-nonspecific, based on sus (CCLE), subacute cutaneous lupus erythematosus
(SCLE) and acute cutaneous lupus erythematosus [3].
*Correspondence:[email protected] Herein we focus on two subsets of CCLE: (1) discoid
1PhiladelphiaVeteransAffairsMedicalCenter,38thandWoodlandAvenues, lupus erythematosus (DLE) and tumid lupus erythema-
Philadelphia,PA08901,USA
tosus (TLE) and (2) SCLE. DLE typically presents as
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scaly, erythematous, disk-shaped patches and plaques, Hospital of the University of Pennsylvania. All subjects
whereas TLE manifests as single or multiple raised poly- who had the diagnosis of DLE, SCLE, TLE and/or DM
cyclic erythematous plaques with a bright reddish or were invited to participate in the study. Seven of the
violaceous smooth surface that does not scar [1]. SCLE DLE patients and one of the SCLE patients had SLE as
typically presents as erythematous papulosquamous, well. All of the recruited patients were receiving therapy
psoriasiform plaques or annular-polycyclic plaques and to treat their disease (see Table 1). Eighteen people
the lesions typically resolve without scarring [4]. Derma- without LE, DM or any autoimmune disease were
tomyositis (DM) is a chronic inflammatory disorder of recruited as controls. To be eligible for the study, con-
the skin and muscles, with histologic findings similar to trols must not have been on corticosteroids or immuno-
SCLE [5,6]. suppressive drugs.
TNFa is a critical proinflammatory cytokine that is Demographic information and data regarding current
implicated in the pathogenesis of multiple inflammatory medications and comorbid illnesses were collected from
diseases. TNFa can be produced by many different cell all subjects. The treating dermatologist evaluated each
types including monocytes, macrophages, dendritic cells, LE and DM patient by performing a complete physical
T and B lymphocytes, natural killer cells, neutrophils, examination and assessing the patient based on Cuta-
mast cells, endothelial cells, fibroblasts and keratinocytes neous Lupus Erythematosus Disease Area and Severity
[7-9]. TNFa is produced as pro-TNFa, which is Index (CLASI) and Systemic Lupus Erythematosus Dis-
expressed on the plasma membrane, where it can be ease Activity Index (SLEDAI) scores for LE patients and
cleaved in the extracellular domain by ADAM17 (also the Cutaneous Dermatomyositis Area and Severity
known as “TACE,” or TNFa-converting enzyme), which Index (CDASI) scores for DM patients to measure dis-
is a matrix metalloproteinase, and results in the release ease activity and damage [22,23]. This study was
of TNFa in a soluble form [10]. ADAM17 mRNA approved by the Institutional Review Board (IRB) at our
expression was found to be increased in lesional skin institution, and all PBMCs were obtained according to
from psoriasis patients [11]. Both membrane-associated the IRB protocol. The Declaration of Helsinki protocols
and soluble TNFa are active [5]. were followed, and patients gave their written, informed
Several studies have examined TNFa in skin lesions consent.
and serum of patients with CLE and DM. TNFa is
increased in lesional skin of patients with DLE and Cell culture and TNFa determination by ELISA
SCLE compared to controls [12-15]. DM lesional skin PBMCs were separated from heparinized venous blood
expresses TNFa, but staining has been found to be of patients with DLE, SCLE, TLE and DM, as well as
more evident in DLE patients and absent in control spe- from controls, on an endotoxin-free Ficoll-Paque
cimens [5]. TNFa was increased in muscle biopsies of PLUS™ gradient (GE Healthcare Bio-Sciences AB,
DM patients [16]. TNFa has been reported to be ele- Uppsala, Sweden). PBMCs were cultured overnight in
vated in sera of some patients with systemic lupus RPMI 1640 medium supplemented with 10% fetal
erythematosus (SLE), DLE and SCLE, but not in that of bovine serum, 1% L-glutamine and 1% penicillin-strep-
patients with DM [17-20]. In vitro production of TNFa tomycin at 37°C and 5% CO . The following morning
2
by unstimulated PBMCs from juvenile DM patients was PBMCs (1 × 105 cells in 0.2 ml per well) were plated in
measured in a previous study in which the investigators 96-well plates (Falcon, Franklin Lakes, NJ, USA) and
found that PBMCs from children who had a disease cultured for 24 hours at 37°C and 5% CO . Conditioned
2
course ≥ 36 months produced more TNFa compared to media were then collected and stored at -80°C. TNFa
PBMCs from juvenile DM patients who had had the dis- was quantitated by ELISA according to the manufac-
ease < 36 months [21]. As there are data regarding turer’s protocol (BD Biosciences, San Diego, CA, USA).
TNFa expression in the skin and sera of CLE and DM All determinations were performed in triplicate. The
patients, but, to the best of our knowledge, minimal viability of cells was measured by trypan blue staining.
data regarding TNFa production from the PBMCs of
these patients, the aim of our study was to evaluate the Flow cytometric analysis
production of TNFa by PBMCs from DLE, SCLE, TLE Prior to intracellular staining for TNFa, PBMCs were
and DM patients. cultured in medium for 4 hours in the presence of bre-
feldin A (1 μg/ml; Invitrogen/Life Technologies, Carls-
Materials and methods bad, CA, USA). To detect intracellular expression of
Subjects TNFa in dendritic cells, approximately 106 PBMCs per
Twenty-one DLE, ten SCLE, ten TLE and eighteen DM sample were stained with lineage 1 cocktail-fluorescein
patients were recruited from the outpatient autoimmu- isothiocyanate (FITC) (lineage cocktail containing anti-
nity clinic in the Department of Dermatology at the bodies against CD3, CD14, CD16, CD19, CD20 and
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Table 1Patient demographics andclinical characteristicsa
Disease Sex(n) Race(n) Meanage,years MeanCLASIorCDASIscore Medications(patients,n)
(range)
DLE(n=21) Females,16 AA,12 45.0(23to64) 8 Antimalarials,18
Males,5 White,8 Methotrexate,2
Hispanic,1 Immunosuppressants,5
Corticosteroids,3
Dapsone,1
Thalidomide,1
SCLE(n=10) Females,7 White,9 47.1(20to64) 9.5 Antimalarials,7
Males,3 Asian,1 Immunosuppressants,2
Corticosteroids,2
Thalidomide,4
TLE(n=10) Females,8 AA,1 42.9(25to57) 10.1 Antimalarials,10
Males,2 White,9 Methotrexate,1
DM(n=18) Females,15 AA,1 49(21to75) 14.7 Antimalarials,10
Males,3 White,16 Methotrexate,6
Asian,1 Immunosuppressants,6
Corticosteroids,9
controls(n=18) Females,10 AA,4 31.2(26to57) N/A N/A
Males,8 White,12
Asian,2
aAA=AfricanAmerican;CDASI=CutaneousDermatomyositisAreaandSeverityIndex;CLASI=CutaneousLupusErythematosusDiseaseAreaandSeverityIndex;
DLE=discoidlupuserythematosus;DM=dermatomyositis;N/A=notapplicable;SCLE=subacutecutaneouslupuserythematosus;TLE=tumidlupus
erythematosus.
CD56), anti-human leukocyte antigen (HLA)-DR-perdi- hoc test were considered significant. Correlations were
nin-chlorophyll-protein (PerCp) and anti-CD123-phy- calculated by performing a Pearson correlation test.
coerythrin (PE) (plasmacytoid dendritic cells (pDCs)) or
anti-CD11c-PE (myeloid dendritic cells (mDCs)) for 30 Results
minutes on ice, followed by 15-minute incubation with Study population
fixation reagent A (FIX & PERM; Invitrogen/Life Tech- The demographic and clinical characteristics of the
nologies). Cells were then washed, permeabilized with study population are summarized in Table 1. Seven of
reagent B (FIX & PERM; Invitrogen Life/Technologies) the DLE patients and one of the SCLE patients had SLE,
and stained with anti-TNFa-allophycocyanin (anti- which did not affect the study results, because removal
TNFa-APC) antibody (or an appropriate isotype con- of these patients from their respective groups had no
trol) for 15 minutes at room temperature. effect on mean values or statistical significance. All of
To detect intracellular expression of TNFa in mono- the recruited patients were receiving therapy to treat
cytes, PBMCs were stained with anti-CD64-FITC and their disease; however, there was no correlation between
anti-CD14-PE followed by anti-TNFa-APC or an appro- the therapy that the patients were receiving and the
priate isotype control as described above. To detect results obtained.
intracellular expression of TNFa in T cells, PBMCs
were stained with anti-CD3-FITC followed by anti- TNFa release from unstimulated DLE PBMCs was
TNFa-APC or an appropriate isotype control as significantly elevated and correlated with disease activity
described above. All antibodies used were purchased Initially, we compared the amount of TNFa released
from BD Biosciences. from PBMCs of nine DLE, five SCLE, five TLE and
Cells were analyzed using a FACSCalibur flow cyt- eight DM patients, as well as eight controls, by ELISA.
ometer (BD Biosciences, San Jose, CA, USA) with BD Compared to controls, DLE PBMCs produced signifi-
CellQuest software (BD Biosciences) at the Flow Cyto- cantly greater amounts of TNFa (P < 0.001) (Figure 1).
metry and Cell Sorting Core, Abramson Cancer Center, However, SCLE, TLE and DM PBMCs did not produce
University of Pennsylvania, Philadelphia, PA, USA. We significantly greater amounts of TNFa compared to
acquired 150,000 events of live cells. controls. The variation of the triplicate of TNFa levels
for each patient was < 10%. The production of TNFa
Statistical analysis was not induced by lipopolysaccharide (LPS) contamina-
One-way analysis of variance followed by Dunnett’s tion, because the maximal level of LPS detected by
multiple comparisons test was used for statistical evalua- Limulus amoebocyte assay (Associates of Cape Cod,
tion as appropriate. P values less than 0.05 in the post East Falmouth, MA, USA) was < 0.015 EU/ml (< 1.5 pg/
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Figure1TNFaproteinproductionbyunstimulatedperipheralbloodmononuclearcellsfromDLE,SCLE,tumidTLEandDMpatients
andfromcontrols.TLEshouldbewritteninthefigurePeripheralbloodmononuclearcells(PBMCs)wereculturedfor24hours,followedby
collectionofsupernatants.TheconcentrationofTNFainthesupernatantswasmeasuredbyELISA.BarsshowthemeansandSD.***P<0.001vs
controlPBMCsbyDunnett’smultiplecomparisontest.DLE=discoidlupuserythematosus,DM=dermatomyositis,TLE,tumidlupus
erythematosus,SCLE,subacutecutaneouslupuserythematosus.
ml), a concentration which gives a negative test result. We observed a trend toward greater amounts of intra-
We also determined the correlation of TNFa release cellular TNFa in monocytes from both DLE and DM
with disease activity in these nine DLE patients using patients compared to controls (P > 0.05) (Figure 4A).
the CLASI score to measure disease activity. A positive There was a statistically significant difference in TNFa
correlation (r = 0.61, P = 0.08) was seen in DLE patients intracellular staining of mDCs when both DLE (P <
between their disease activity and TNFa protein secre- 0.001) and DM (P < 0.001) patients were compared to
tion (Figure 2). There was no correlation seen in SCLE controls (Figure 4B).
(r = -0.05) or DM (r = 0.3493) patients between their
disease activity and TNFa protein secretion. Mean fluorescence intensity of TNFa release by mDCs
and monocytes
Myeloid dendritic cells expressing intracellular TNFa were The mean fluorescence intensity (MFI) of TNFa release
significantly greater in DLE and DM patients than in fromCD64+CD14+monocytesandCD11c+HLA-DRhi/low
controls mDCs was measured among five DLE and five DM
The dot plots shown in Figure 3 demonstrate popula- patients, as well as five controls. There was a 3.3-fold
tions of both mDCs (Figure 3A) and monocytes (Figure increase in the MFI of monocytes (P < 0.01) and a 3.6-
3B) from representative individuals, one from each fold increase in the MFI of mDCs (P < 0.01) from DLE
group, who were defined and/or gated by antibodies patients compared to controls (Figure 5). There was no
(upper panel) and subsequently analyzed for their ability significant difference between the MFIs of both mono-
to produce TNFa (lower panel). cytesandmDCsfromDMpatientscomparedtocontrols.
We determined the percentage of CD64+CD14+
monocytes, CD123+ pDCs, CD11c+HLA-DRhi/low mDCs CD64+CD14+ monocytes and CD11c+HLA-DRhi/low myeloid
and CD3+ T lymphocytes expressing intracellular TNFa dendritic cells were not significantly greater in DLE
in five DLE, five DM patients and five controls. The patients
results from all patients demonstrated that major produ- The size of the monocyte and mDC populations was
cers of TNFa were monocytes and mDCs (Figure 4). measured in five DLE and five DM patients, as well as
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Figure2TNFainperipheralbloodmononuclearcellsupernatantsfromdiscoidlupuserythematosus(DLE)patientscorrelatedwith
CutaneousLupusErythematosusDiseaseAreaandSeverityIndexscores(r=0.61).
in five controls. The size of the CD64+CD14+ monocyte intracellular staining from mDCs of DLE patients (P <
population in DLE patients was 1.7-fold greater com- 0.001) and DM patients (P < 0.001) compared to con-
pared to DM patients (P > 0.05) and 2.2-fold greater trols. A significantly greater MFI from monocytes and
compared to controls (P > 0.05) (Figure 6A). The size of mDCs from DLE patients compared to controls was also
the CD11c+HLA-DRhi/low mDC population of DLE present, whereas there was no statistically significant dif-
patients was 1.4-fold greater compared to that of DM ference in MFI from monocytes and mDCs between DM
patients (P > 0.05) and 3.1-fold greater than that of con- patients and controls. The significant elevation of the
trols (P > 0.05) (Figure 6B). MFI in DLE patients compared to controls demonstrates
that the monocytes and mDCs from DLE patients are
Discussion synthesizing TNFa in greater quantities compared to
The proinflammatory nature of TNFa in inflammatory controls. The MFI of the PBMCs from DLE patients is
diseases is well-established (reviewed in [7]). TNFa significantly greater than that of controls. Our ELISA
causes the upregulation of adhesion molecules and che- results demonstrate an approximately 15-fold increase
mokines, which leads to attachment of inflammatory in TNFa release from DLE PBMCs compared to those
cells to vessels, rolling, emigration and eventually che- seen in DM, and the flow cytometry results explain this
motaxis [24-26]. TNFa is an activating cytokine for finding in part. We realize the monocytes and mDCs
fibroblasts and leukocytes, including dendritic cells. comprise a relatively small percentage of circulating
TNFa stimulation leads to increased production of IFN- cells and that there may be another population of cells
g, a cytokine that is also increased in lesional skin of that we did not stain that could contribute to the
DLE patients [12]. Our study demonstrates that TNFa increased TNFa release in DLE. We will investigate
release is significantly greater in PBMCs from DLE other cell types in the future.
patients compared to controls. We next explored whether there were any differences
We next determined which specific cell type was between the size of the monocyte and mDC populations
secreting the increased TNFa in DLE patients. Our flow amongtheDLEandDMpatientsandcontrols.Wefound
cytometry results demonstrated that the majority of a trend toward an increased population of monocytes in
TNFa was produced by monocytes and mDCs, with a DLEpatientscomparedtobothDMpatientsandcontrols.
statistically significant difference between TNFa The mDC population was trending toward greater
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Figure3 Flow cytometric analysis of intracellular TNFa production bymyeloid dendritic cells and monocytes from patients and
controls.Upperpanelsshowgatingofperipheralbloodmononuclearcells(PBMCs)fromrepresentativepatientswithdiscoidlupus
erythematosus(DLE),dermatomyositis(DM)orcontrolsstainedwithfluorochrome-conjugatedappropriateantibodies(A)myeloiddendriticcells
(lineagenegative,HLADR+,CD11c+)or(B)monocytes(CD64+CD14+).LowerpanelsshowintracellularexpressionofTNFain(A)myeloid
dendriticcellsor(B)monocytes.ThenumbersintheupperpanelsstatethepercentageofcellsubsetpertotalpopulationofPBMCs.The
numbersinthelowerpanelsstatethepercentageofmyeloiddendriticcellsormonocytesproducingTNFa.
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A. B.
CD64+CD14+Monocytes CD11c+HLA-DRhi/low mDCs
***
Staining 60 Staining 4500 ***
(cid:68) 40 (cid:68)
NF NF 30
T T
acellular 20 cellular 1200
ntr ntr
% I 0 % I 0
Normal DLE DM Normal DLE DM
C. D.
CD123+ pDCs CD3+ T Cells
ng 8 ng 0.6
ni ni
ai ai
St 6 St
(cid:68) (cid:68) 0.4
F F
N N
T 4 T
ar ar
ul ul 0.2
ell 2 ell
c c
r r
nt nt
% I 0 % I 0.0
Normal DLE DM Normal DLE DM
Figure4Definingthecellpopulation responsibleforTNFaproduction.(A) CD64+CD14+monocytes,(B) CD11c+HLA-DRhi/lowmyeloid
dendriticcells,(C)CD123+plasmacytoiddendriticcellsand(D)CD3+Tlymphocytesfromfivediscoidlupuserythematosus(DLE)andfive
dermatomyositis(DM)patients,aswellasfromfivecontrols,wereanalyzedbyflowcytometrytoassesstheirabilitytoproduceintracellular
TNFa.BarsshowthemeansandSD.***P<0.001vscontrolsbyDunnett’smultiplecomparisonstest.
numbers in DLE patients compared to controls. These elevated levels of IFNa and IFNb (both type I IFNs)
flowcytometryresultssupportourculturedPBMCELISA [27,28]. Because pDCs are the main cell type producing
results,whichshowedthattherewasasignificantlygreater IFNa and IFNb, they have been implicated in disease
amount of TNFa released from DLE patients. The pathogenesis. A higher proportion of pDCs are also pre-
increasedamountofTNFainmonocytesandmDCsfrom sent in the skin than in the blood of patients with SLE
DLEpatientsandthegreatersizeofthesecellpopulations and CLE, suggesting that pDCs migrate from the circu-
in DLE patients may further explain why PBMCs from lation to the skin and may play a role in the production
DLEpatientsreleasegreaterquantitiesofTNFa. of skin lesions [29,30]. Our experiments demonstrate
Plasmacytoid DCs have been implicated in the patho- that TNFa levels produced by PBMCs vary with the dif-
genesis of both SLE and CLE. SLE is characterized by ferent CLE subsets and with DM. We found that pDCs
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A. B. CD11c+HLA-DRhi/low mDCs
CD64+CD14+Monocytes
**
4000 ** 1500
3000
1000
FI 2000 FI
M M
500
1000
0 0
Normal DLE DM Normal DLE DM
C. D.
CD3+ T Cells
CD123+ pDCs
8000 2000
6000 1500
FI 4000 FI 1000
M M
2000 500
0 0
Normal DLE DM Normal DLE DM
Figure5Measurementofmeanfluorescenceintensity(MFI).TheMFIofTNFareleasefrom(A)CD64+CD14+monocytes,(B)CD11c+HLA-
DRhi/lowmyeloiddendriticcells,(C)CD123+plasmacytoiddendriticcells,and(D)CD3+Tlymphocytesfromfivediscoidlupuserythematosus
(DLE)andfivedermatomyositis(DM)patients,aswellasfromfivecontrols,asdeterminedbyflowcytometry.BarsshowthemeansandSD.**P
<0.01vscontrolsbyDunnett’smultiplecomparisontest.
produced minimal amounts of TNFa compared to producing dendritic cells) has been found in elevated
monocytes and mDCs. Therefore, we postulate that amounts in psoriasis plaques compared with nonlesional
monocytes and mDCs may have a bigger role in the skin from psoriasis patients and normal skin [9,31].
pathogenesis of DLE than was originally believed. The These Tip-DCs are CD11c+, BDCA-1- and HLA-DR+
increased numbers of inflammatory cells seen in the [31]. Tip-DCs express HLA-DR at 10-fold lower levels
skin of DLE patients relative to DM patients and con- compared to CD11c+ and BDCA-1+ mDCs. It is possible
trols may be explained, at least in part, by the increased that some of the CD11c+HLA-DRlow mDCs in our DLE
TNFa produced in DLE, given the importance of TNFa patients might actually have been Tip-DCs. It has been
in cell recruitment to endothelial cells and skin [25]. shown that bone marrow-derived monocytes accumulate
Flow cytometry showed that both CD11c+HLA-DRhi and differentiate to Tip-DCs in the spleen, liver and
and CD11c+HLA-DRlow mDCs produced TNFa and lymph nodes of Trypanosoma brucei brucei-infected
accounted for much of the TNFa production exhibited mice [32]. Another type of inflammatory mDCs, known
by PBMCs. Recently, an inflammatory mDC known as as M-DC8+ cells, has also recently been described that
Tip-DC (TNFa-inducible nitric oxide synthase- are CD11c+, CD14+, CD16+, CD64- and HLA-DR+
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A.
CD64+CD14+Monocytes
20
s 15
ll
e
C
al
t 10
o
T
f
o
% 5
0
Normal DLE DM
B.
CD11c+HLA-DRhi/low mDCs
3
s
ell 2
C
al
t
o
T
of 1
%
0
Normal DLE DM
Figure6Measurementofcellpopulationsbyflowcytometry.(A)CD64+CD14+monocyteand(B)CD11c+HLA-DRhi/lowmyeloiddendritic
cellpopulationsfromfivediscoidlupuserythematosus(DLE)andfivedermatomyositis(DM)patients,aswellasfromfivecontrols,as
determinedbyflowcytometry.
[33,34]. These M-DC8+ cells are able to produce signifi- monocytes and CD11c+HLA-DRhi/low mDCs. The MFIs
cantly greater amounts of TNFa in response to LPS of the monocyte and mDC populations are also
compared to M-DC8- monocytes and DCs [8,35]. increased in DLE, suggesting that these cells likely have
Although we have not directly addressed these two a greater role in DLE than originally believed. Future
populations of mDCs in our current studies, there is a studies using surface staining for Tip-DCs and M-DC8+
possibility that these populations might contribute to mDCs by flow cytometry will allow us to explore the
enhanced TNFa production in DLE patients. Further role of these cell populations in DLE.
studies designed to determine a potential role of these
predominantly tissue-based DCs are being performed.
Abbreviations
CCLE:chroniccutaneouslupuserythematosus;CDASI:Cutaneous
Conclusions DermatomyositisAreaandSeverityIndex;CLASI:CutaneousLupus
Our findings suggest that DLE is associated with ErythematosusDiseaseAreaandSeverityIndex;CLE:cutaneouslupus
increased TNFa protein production from CD64+CD14+ erythematosus;DLE:discoidlupuserythematosus;DM:dermatomyositis;
ELISA:enzyme-linkedimmunosorbentassay;IFN:interferon;mDC:myeloid
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dendriticcell;PBMC:peripheralbloodmononuclearcell;RT-PCR:real-time 11. KawaguchiM,MitsuhashiY,KondoS:Overexpressionoftumournecrosis
polymerasechainreaction;SCLE:subacutecutaneouslupuserythematosus; factor-α-convertingenzymeinpsoriasis.BrJDermatol2005,152:915-919.
SLE:systemiclupuserythematosus;SLEDAI:SystemicLupusErythematosus 12. ToroJR,FinlayD,DouX,ZhengSC,LeBoitPE,ConnollyMK:Detectionof
DiseaseActivityIndex;TLE:tumidlupuserythematosus;TNFα:tumornecrosis type1cytokinesindiscoidlupuserythematosus.ArchDermatol2000,
factorα. 136:1497-1501.
13. PopovicK,EkM,EspinosaA,PadyukovL,HarrisHE,Wahren-HerleniusM,
Acknowledgements NybergF:Increasedexpressionofthenovelproinflammatorycytokine
Thisstudywassupportedinpartbyameritreviewgrantfromthe highmobilitygroupboxchromosomalprotein1inskinlesionsof
DepartmentofVeteransAffairs,VeteransHealthAdministration,Officeof patientswithlupuserthematosus.ArthrRheum2005,52:3639-3645.
ResearchandDevelopment,BiomedicalLaboratoryResearchand 14. DroseraM,FacchettiF,LandolfoS,MondiniM,NybergF,ParodiA,
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(toVPW).).Inaddition,fundingwasprovidedbytheDepartmentofVeterans inthepathogenesisofautoimmuneskindiseases.ClinExpRheumatol
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Medicine,UniversityofPennsylvania,2MaloneyBuilding,36thandSpruce tumornecrosisfactor-αanditsreceptorsininflammatorymyopathies.
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18. SabryA,SheashaaH,El-HusseiniA,MahmoudK,EldahshanKF,GeorgeSK,
Authors’contributions Abdel-KhalekE,El-ShafeyEM,Abo-ZenahH:Proinflammatorycytokines
ASNandMMBcarriedoutthePBMCculturesandTNFELISAstudies.ABand (TNF-αandIL-6)inEgyptianpatientswithSLE:itscorrelationwith
MWparticipatedintheflowstudies,andABdraftedthemanuscript.VPW diseaseactivity.Cytokine2006,35:148-153.
performedtheCLASIassessments.VPWandASNparticipatedinthedesign 19. MaczynskaI,MilloB,Ratajczak-StefańskaV,MaleszkaR,SzychZ,KurpiszM,
ofthestudy.MSperformedthestatisticalanalysis.VPWconceivedthestudy, Giedrys-KalembaS:Proinflammatorycytokine(IL-1β,IL-6,IL-12,IL-18and
participatedinitsdesignandcoordinationandhelpedtodraftthe TNF-α)levelsinseraofpatientswithsubacutecutaneouslupus
manuscript.Allauthorsreadandapprovedthefinalmanuscript. erythematosus(SCLE).ImmunolLett2006,102:79-82.
20. ShimizuT,TomitaY,SonK,NishinaritaS,SawadaS,HorieT:Elevationof
Competinginterests serumsolubletumournecrosisfactorreceptorsinpatientswith
Theauthorsdeclarethattheyhavenocompetinginterests. polymyositisanddermatomyositis.ClinRheum2000,19:352-359.
21. PachmanLM,Liotta-DavisMR,HongDK,KinsellaTR,MendezEP,KinderJM,
Received:22November2010 Revised:21September2011 ChenEH:TNFα-308Aalleleinjuveniledermatomyositis:associationwith
Accepted:4January2012 Published:4January2012 increasedproductionoftumornecrosisfactorα,diseaseduration,and
pathologiccalcifications.ArthritisRheum2000,43:2368-2377.
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