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Original Article
Apoptosis in chronic cutaneous lupus erythematosus,
discoid lupus, and lupus profundus
Claudia Ileana Senz-Corral1, Mara Elisa Vega-Memje1, Eduwiges Martnez-Luna1, Juan Carlos
Cuevas-Gonzlez2, Alma Anglica Rodrguez-Carren*, Juan Jos Bollain-y-Goytia de la Rosa3, Felipe de
Jess Torres del Muro3, Esperanza Avalos-Daz3
1
Department of Dermatology, Dr. Manuel Gea Gonzlez General Hospital, Mxico City, Mexico; 2Pathology
Laboratory, Faculty of Dentistry, Juarez University of The State of Durango, Durango, Dgo, Mexico; 3Laboratory
of Immunology and Molecular Biology, UABE, Universidad Autnoma de Zacatecas, Zacatecas, Zac, Mexico.
*
Professor of Medical School ITESM Campus Guadalajara Mexico. Private Practice (Dermocare).
Received March 30, 2015; Accepted May 21, 2015; Epub June 1, 2015; Published June 15, 2015
Keywords: Apoptosis, discoid lupus, lupus profundus, chronic cutaneous lupus erythematosus, proapoptotic mol-
ecules
DLE is the most common type of CLE, affecting In most frequent dermatological consultations
the face, scalp, ears, neck, and the extensor of CLE cases correspond to DLE and LP, both
surfaces of thoracic limbs [6]. DLE occurs in dermatologic features display lower morbidity
women of reproductive age (20-40 years), rang- and mortality that the SLE, in this the quality of
Apoptosis in lupus
life is practically limited to physical ap- The study groups comprised 10 cases of LP
pearance. and 10 cases of DLE, and a control (healthy tis-
sue without histological alteration).
Five percent of patients with DLE progress to
SLE that tends to be less aggressive than in Skin samples of cases and controls were pro-
those in whom systemic disease begins abrupt- cessed for immunohistochemistry (IHC). Two-
ly [9, 10]. micrometer-thick sections were deparaffinized
for 10 minutes at 60C and hydrated in xylene
This condition develops in genetically suscepti- and an alcohol series (100%, 95%, and 70%).
ble subjects who are exposed to environmental After being washed with 1X phosphate-buff-
factors, such as UV radiation, infection, and ered saline (PBS) for 5 minutes, they were heat-
drugs, which accelerate progression of the dis- ed for 1 minute in citrate buffer, pH 6 for anti-
ease [11]. gen retrieval, and endogenous peroxidase was
blocked for 10 minutes with 3% H2O2 dissolved
The clinical presentation consists of erythema,
in metanol.
hyper- or hypopigmentation, plaques, and atro-
phy [4, 5]. LP is characterized by an inflamma- The tissues were incubated for 18 hours at 4C
tory infiltrate in the subcutaneous tissue and in a moist chamber with monoclonal anti-Fas-L.
deep dermis, manifesting clinically as erythe- (RDI-CD95Lamb. RDI Flanders, NJ, USA), anti-
ma with subcutaneous nodules and an ery- body -Fas (DAKO) and anti-Bax (RDI-BAX-
thematous surface or fovea when the evolution amb-A7 RDI Flanders, NJ, USA), diluted 1:100.
is chronic. Then, the sections were incubated with HRP-
conjugated anti-mouse IgG (Cat. 50695148
Local skin damage due to physical, chemical,
Zymed San Francisco, California) for 4 h and
and biological factors (eg, environmental, infec-
washed twice with PBS for 5 minutes. The color
tious, hormonal, and stress-related) induces
reaction was induced by 3,3-diaminobenzi-
apoptosis, in which antigens and intracellular
dine-0.06% H2O2 (Sigma, St Louis, MO), and the
molecules translocate in the cell surface [6].
reaction was stopped with 0.5 M sulfuric acid,
Apoptosis is an active form of programmed cell
and counterstained with hematoxylin for 1 min-
death in response to external or internal molec-
ute. After a dehydration step in alcohol, the
ular signals and is detected directly and indi-
specimens were cleared with xylene and
rectly. TUNEL (transferase-mediated dUTP nick-
mounted in synthetic resin for microscopy
end labeling) is a direct method, whereas
(Olympus B-Max BX-40) at 20X and 40X.
immunohistochemistry is an indirect approach,
in which the expression of molecules that regu- The analysis was performed by 2 investigators
late apoptosis is analyzed, such as Fas, Fas-L, whom examined the tissues in a blinded man-
Bax, Bcl-2, and p53 [12]. ner, ten random fields were tested to determine
the bit rates of immunoreactivity. The images
The aim of this study was to analyze the expres-
were analyzed with Image-Pro Plus, Version 7.0
sion of proapoptotic molecules in patients with
for WindowsTM.
lupus erythematosus discoid and lupus profun-
dus, this is of importance because its clinical
Apoptosis was measured by DNA fragmenta-
implication in skin regeneration and reparative
tion with an apoptosis detection kit (Roche
process in lupus profundus.
TUNEL-Apo-AP; Cat 11684795910 Penzberg,
Material and methods Germany). Tissues were deparaffinized and
hydrated as in the IHC experiments. After being
This descriptive study was conducted at the washed in PBS for 5 minutes, the sections were
Department of Dermatology of General Hospital incubated with TUNEL mixture for 1 hour at
Dr. Manuel Gea Gonzlez and in the 37C in the dark.
Department of Immunology of the Universidad
Autnoma de Zacatecas. (Approved by the eth- The samples were washed in PBS and mounted
ics committee and research of the General in fluorescence mounting medium (Dako, Cat.
Hospital Dr. Manuel Gea Gonzlez #06-40- S3023, Dakocytomation), for analysis at 20X
2011). Patients signed informed consent for and 40X under a fluorescence microscope
diagnosis and treatment. (Olympus B-Max BX-40).
To distinguish apoptotic and nonapoptotic sam- compared with controls. We also noted differ-
ples, cells were counterstained with propidium ences in Fas-L, -Fas, and -Bax proteins expres-
iodide. As a positive control for the TUNEL stain- sion intensity in DLE patients in the epidermis,
ing, a sample was treated with DNase-1 for 30 and hair follicles, as well as long inflammatory
minutes, and the negative control was incubat- infiltrates of DLE and other groups of lupus (X2
ed with labeling solution without enzyme. = 11.000 P = 0.027) (Table 1).
Figure 1. TUNEL staining of lupus profundus (400 ). A. Positivity in the epidermis. B. Reaction in the hair follicle and
adjacent lymphocytic infiltrate. C. Control. D. Control +.
Apoptosis is physiologically programmed cell Fas receptor triggers apoptosis [17] and has
death that can be initiated by a permanently other nonapoptotic signaling functions that are
active mechanism (extrinsic pathway) or by poorly understood, such as in extracellular sig-
irreparable DNA damage due to external fac- naling, invasion, and cell proliferation [18].
tors (intrinsic pathway).
Based in our findings, the Fas-L and its Fas
Apoptosis begins in the membrane with a sig- receptor are expressed at similar levels in LP
nal that is induced by cellular damage and by and DLE (mild to moderate), our findings are
the binding of ligands to their receptors and is consistent with other authors that demonstrat-
characterized by the loss of membrane integri- ed increased activity of the extrinsic apoptotic
ty [14, 15]. pathway.
The extrinsic pathway of apoptosis is effected Bax is a proapoptotic member of the Bcl-2 fam-
by cytokine receptors and ligands from the ily that regulates intrinsic apoptotic signaling
tumor necrosis factor family. Fas-L and Fas [19]. Bax is expressed in the cytoplasm in an
receptor mediate immune regulation and the inactive form and is activated in the early stag-
development of autoimmunity, and mutations es of apoptosis. It is associated with mitochon-
in Fas and its ligand cause severe lymphade- dria through poorly understood mechanisms
nopathy and autoimmunity in humans and and induces conformational changes [20].
mice. The function of Fas-L is to stimulate Fas
receptor on the cell membrane, initiating signal Ohsako et al. studied SLE activity, reporting sig-
transduction and activating caspases [16]. nificant expression of Bcl2 and Bax in cases of
active versus inactive disease. This group also Address correspondence to: Dr. Mara Elisa Vega
discussed the hypothesis of Oltvai, in which Memje, Department of Dermatology, Dr. Manuel
overexpression of Bax accelerates cell death Gea Gonzlez General Hospital Calzada de Tlalpan
and proposed that the anti-Bcl2 and -Bax can 4800, Seccin XVI Delegacin Tlalpan, Mxico, D.F.
be used as indicators of the rate of programmed C.P 14080. Tel: 4000-3000; Fax: 4000-3000;
cell death [21]. E-mail: elisavega50@gmail.com
[13] Navarrete CL, Ibez C. Rol de la apoptosis en [19] Zarnescu O, Brehar FM, Chivu M, Ciurea AV.
la fisiopatologa del lupus eritematoso sistemi- Immunohistochemical localization of cas-
co. Rev Chil Reumatol 2008; 24: 30-38. pase-3, caspase-9 and Bax in U87 glioblasto-
[14] Evan G, Littlewood T. A matter of life and cell ma xenografts. J Mol Histol 2008; 39: 561-9.
death. Science 1998; 281: 1317-22. [20] Lalier L, Cartron PF, Olivier C, Log C, Bougras
[15] Alfaro ME, Garca CC, Dueas GA. Mtodos de G, Robert JM, Oliver L, Vallette FM.
deteccin de la apoptosis, aplicaciones y limi- Prostaglandins antagonistically control Bax ac-
taciones. Rev Inst Nal Cancerol Mx 2000; 46: tivation during apoptosis. Cell Death Differ
275-80. 2011; 18: 528-37.
[16] O Reilly LA, Tai L, Lee L, Kruse EA, Grabow S, [21] Ohsako S, Hara M, Harigai M, Fukasawa C,
Fairlie WD, Haynes NM, Tarlinton DM, Zhang Krajewski S, Reed J, Kashiwazaki S. The Bcl-2/
JG, Belz GT, Smyth MJ, Bouillet P, Robb L, Bax ratio of lymphocytes from human systemic
Strasser A. Membrane-bound Fas ligand only lupus erythematosus patients. Japanese J
is essential for Fas-induced apoptosis. Nature Rheumatol 1997; 7: 305-313.
2009; 461: 659-63. [22] Faurschou M, Penkowa M, Andersen CB,
[17] Phil Dash. Apoptosis; Basic Medical Sciences, Starklint H, Jacobsen S. Renal cell apoptosis in
St.Georges, University of London; 1994. human lupus nephritis: a histological study.
http://www.ppgorgsistem.ics.ufba.br/arquiv- Lupus 2009; 18: 994-9.
os/fatima/Apoptosis.pdf.
[18] Brint E, OCallaghan G, Houston A. Life in the
Fas lane: differential outcomes of Fas signal-
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