ARTICLE
Auteur(s) : S Nur
Aksoy1, Zulal Erbagci2, E Ilker
Saygili3, Tugce Sever4, A Binnur
Erbagci1, Sacide Pehlivan4
1Department of Biochemistry, Medical Faculty
of Gaziantep University, 27310, Gaziantep, Turkey
2Department of Dermatology, Medical Faculty
of Gaziantep University
3Vocational School of Higher Education
for Health Services, Gaziantep University
4Department of Medical Biology, Medical Faculty
of Gaziantep University
accepté le 24 Juillet 2009
Vitiligo is a chronic disease of the skin, characterized by loss
of melanin pigment and functional melanocytes. The etiopathogenesis
of vitiligo has not yet been clearly identified. Among various
explanations there are the autoimmune theory, autocytotoxic theory
and neural theory, all of which are unable to explain the entire
pathophysiology [1]. In recent years, there has been convincing
evidence about the presence of imbalance in oxidative status in
vitiligo [2-15]. It has been reported that hydrogen peroxide
accumulation occurs in vitiligo due to impairment in the
oxidant/antioxidant balance [14, 15].
Myeloperoxidase (MPO; EC 1.11.1.7) is a heme containing
peroxidase that is abundantly expressed in the azurophilic granules
of neutrophils and to a lesser extent in monocytes [16]. Altered
levels and/or production of proinflammatory cytokines, IL-6, TNF-α,
and the neutrophile chemoattractant cytokine, IL-8, may indicate a
possible role of neutrophiles in the pathogenesis of vitiligo
[17-19]. MPO catalyses the formation of hypochloric acid (HOCI)
from the chlor ion and hydrogen peroxide. HOCI is the potent
antimicrobial oxidant formed in the active neutrophiles [16, 20,
21]. HOCI might increase the formation of hydroxil radicals by both
Fe+3 dependent and independent pathways [22, 23].
MPO is coded by a single gene formed by 11 introns and 12 exons
of 11 kb and localized to the long arm of the 17th
chromosome and its expression is limited by myeloid cells [24].
During granulocyte differentiation in bone marrow, only
promyelocytes and promyelomonocytes actively produce MPO (25). In
tissue, MPO gene expression and differentiation are regulated by
various transcription factors and MPO synthesis halts in the
myeloid phase of differentiation [25-27]. The MPO gene is expressed
specifically in immature myeloid cells, and its expression is
tightly regulated [28]. GM-CSF is known as a priming agent for
neutrophil MPO activity [29, 30].
To our knowledge, there is no study in the literature regarding
MPO enzyme activity and gene polymorphism in patients with
vitiligo. In the present study, we aimed to investigate the role of
MPO in melanin synthesis by means of MPO enzyme activity and gene
polymorphism.
Materıals and methods
Materials
The study protocol conforms to the principles of the Helsinki
Declaration and the institutional ethics review board approved it.
All participants were given informed consent. 54 patients with the
diagnosis of vitiligo (30 females (55%), 24 males (45%)) and age
and sex matched healthy controls who were admitted to the
Dermatology Department of Gaziantep University Hospital were
included into the study. After excluding plasma with lipemia and
hemolysis, 50 healthy controls (23 females (46%), 27 males (54%))
for MPO enzyme activity and 58 healthy controls (31 females (53%),
27 males (47%)) for gene polymorphism were included as control
groups. Subjects with diabetes mellitus, hypertension,
cardiovascular disorders, autoimmune diseases or those with chronic
drug use for any other disease were not included into the study.
Demographic characteristics and whole blood counts of the
participants were recorded. Subjects with vitiligo were divided
into three groups (generalized, acrofacial and local) according to
the localization of depigmented macules.
For MPO analysis, 4 mL of venous blood was drawn into tubes
with lithium heparin and centrifuged within 4 hours at 3000×g
for 10 minutes at 4 ◦C. Plasma samples were stored
at – 70 ◦C. For DNA analysis, venous blood was
obtained from the tubes with Ethylene Diamine Tetra Acetic Acid
(EDTA) and stored at – 20 ◦C.
Methods
MPO Enzyme Levels
Plasma MPO activity was measured with the ELISA method (Oxford
Biomedical Research, Inc., USA). The MPO-EIA assay system is a
“sandwich” ELISA. Antigens captured by a solid phase monoclonal
antibody were detected with an HRP-labeled Mouse monoclonal
anti-MPO. The HRP substrate TMB was added, and a blue color
developed as the reaction proceeded. The reaction was stopped with
the addition of the stop solution, and the yellow product was
measured at 405 nm.
DNA isolation and PCR
Genomic DNA was extracted from whole blood samples with the use of
Puregene DNA purification kits (Puregene, Gentra, USA). Genotyping
was performed by PCR-RFLP method [31]. The region with a -463
polymorphism, which is located at the promotor of the MPO gene, was
amplified with PCR by using MPOF (5’ CGG TAT AGG CAC AAT GGT GAG)
and MPOR (5’ GCA ATG GTT CAA GCG ATT CTT C) primer pair and
amplification control was done with 2% agarose gel. 1.5 mmol/L
MgCl2 was used for 10 umol/L amplification of each
primary and 0.2 mmol/L and 25 uL amplification of each
dNTP (A, G, T and C). After initial heating at
95 oC for 5 min, 31 PCR cycles were
performed and consisted of heat denaturation (95 oC
for 45 s), annealing (57.3 oC for 45 s)
and extension (72 oC for 1 min). A final
extension (72 oC for 7 min) was performed. The
amplified region was incubated for 16 hours with 5 units of AciI
enzyme at 37 oC and analysed with 3% agarose gel
(figure 1).
Statistical analyses
Median (min-max) values were calculated for continuous variables
and absolute and relative frequencies for categorical variables.
Because MPO levels were highly skewed, logarithmic transformation
was performed before analysis. The unpaired student T test and
one-way ANOVA were used for comparing the groups and subgroups
respectively for continuous variables. Genotypes and allele
distributions were controlled with chi-square test and
Hardy-Weinberg equilibrium
(http://ihg2.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl). Two tailed
P-values less than 0.05 were defined as significant. Statistical
analyses were performed using SPSS for Windows software version
11.0 (SPSS Inc. Chicago, IL, USA).
Results
The demographic characteristics of the patients and control group
are presented in table 1. The mean age
of the patients with vitiligo was 28, ranging between 7 and 63. The
mean age of the control group was 33, ranging between 6 and 63. 10%
of the patients and 14% of the control group were smokers. The
groups were comparable in terms of age, sex, smoking ratio, body
mass index, leucocyte and neutrophile count. There was no
significant difference between the groups in terms of MPO genotype
and allele frequency (table 2). No
significant deviation from the Hardy-Weinberg equilibrium was found
for the alleles in patients and controls (HWE-Control p: 0.678,
HWE-Vitiligo p: 0.297).
Plasma MPO enzyme levels were 60 (10-180) ng/mL in patients and
125 (20-230) ng/mL in the control group (p = 0.005),
respectively (table 1). Subjects with
vitiligo and the control group were also evaluated according to
genotype distribution (table 3). In
subjects with vitiligo, there was no significant difference in MPO
levels between the groups with GG (60(10-180) ng/mL) or GA
(55(10-180) ng/mL) polymorphism (p = 0.9). In subjects with
vitiligo, the MPO level in the group with an AA genotype was
82 ng/mL. In the control group, there was no significant
difference in MPO levels between the groups with GG
(130(20-240) ng/mL) or GA (105(20-220) ng/mL)
polymorphism (p = 0.4). In the control group, the MPO level in the
group with an AA genotype was 220 ng/mL (table 3).
MPO enzyme activity was similar in the generalized
(65(10-180) ng/mL), acrofacial (80(10-180) ng/mL), and local
(40(25-92) ng/mL) subtypes of vitiligo (p = 0.8) (table 4).
Table 1 Demographic characteristics of the patients
with vitiligo and the control group
|
Vitiligo n = 54
|
Control n = 50
|
P-value
|
|
Age (year)
|
28 (7-63)
|
33 (6-63)
|
0.1
|
|
Sex (Female/Male)
|
30/24
|
23/27
|
0.1
|
|
Smoking (%)
|
10
|
14
|
0.5
|
|
Body-Mass Index
|
23.05 ± 3.03
|
24.9 ± 4.7
|
0.1
|
|
Leucocyte
|
7069 ± 1555
|
6977 ± 1336
|
0.7
|
|
Neutrophil
|
3928 ± 1354
|
3831 ± 1054
|
0.6
|
|
Hemoglobin (gr/dL)
|
13.83 ± 1.41
|
14.26 ± 1.36
|
0.1
|
|
MPO (ng/mL)*
|
60 (10-180)
|
125 (20-230)
|
0.005
|
Table 2 Comparison of MPO (-463) gene polymorphism
frequency between patients with vitiligo and healthy controls (NS:
nonsignificant)
|
MPO (-463)
|
Control N %
|
Vitiligo N %
|
P-Value
|
|
Genotypes
|
|
|
|
|
GG
|
37 (63.8)
|
33 (61.1)
|
NS
|
|
AG
|
18 (31.0)
|
20 (37.0)
|
NS
|
|
AA
|
3 (5.2)
|
1 (1.9)
|
NS
|
|
Sum
|
58 (100.0)
|
54 (100.0)
|
|
|
Allelles
|
|
|
|
|
A
|
24 (20.7)
|
22 (20.3)
|
NS
|
|
G
|
92 (79.3)
|
86 (79.6)
|
NS
|
|
Sum
|
116 (100.0)
|
108 (100.0)
|
|
Table 3 Comparison of genotypes and enzyme levels of
MPO in patients with vitiligo and the control group
|
Vitiligo
|
Control
|
|
MPO-463
|
n
|
MPO*
|
n
|
MPO*
|
|
Genotype
|
|
|
|
|
|
GG
|
33
|
60 (10-180)
|
35
|
130 (20-240)
|
|
GA
|
20
|
55 (10-180)
|
14
|
105 (20-220)
|
|
AA
|
1
|
82
|
1
|
220
|
|
Sum
|
54
|
|
50
|
|
Table 4 Vitiligo types and MPO enzyme activity
|
Vitiligo type
|
N
|
MPO*
|
|
Generalized
|
26
|
65 (10-180)
|
|
Acrofacial
|
14
|
80 (10-180)
|
|
Local
|
14
|
40 (25-92)
|
Discussıon
Vitiligo is an acquired, idiopathic, depigmenting disorder
characterized by sharply defined white macules, resulting from the
loss of melanocytes from the cutaneous epidermis [32]. There are
several reports stating that vitiligo is associated with increased
oxidative stress in the entire epidermis [33]. It has been
demonstrated that epidermal hydrogen peroxide (H2O2) is increased
and catalase is decreased in subjects with vitiligo. These findings
indicate the presence of major stress, due to increased epidermal
H2O2. Therefore, hydroxil radical affects melanin and causes
membrane damage via lipid peroxidation reactions [2, 34, 35].
The presence of various cytokines has been analysed in the
autoimmune mechanisms of vitiligo. IL-6, which is produced by
mononuclear cells, was found to be increased in vitiligo [18]. This
cytokine can induce the expression of intercellular adhesion
molecule 1 (ICAM-1) in melanocytes and facilitate the
leucocyte-melanocyte interaction, resulting in immunological damage
in pigment cells [18, 36]. It has previously been reported that
IL-8 production is increased in mononuclear cells of patients with
vitiligo. IL-8 might attract neutrophils to the sites of lesions in
vitiligo, resulting in augmented inflammation and melanocyte damage
[18]. GM-CSF has been reported to function as an intrinsic growth
factor for melanocytes [37]. Tu et al. reported increased
serum GM-CSF levels in both focal type and generalized type
vitiligo. Moreover, serum GM-CSF levels in the progressive stage
were higher than those in stable stages. However, this seems
controversial, with the results of the study of Yu et al., who
reported a decrease in the production of GM-CSF by mononuclear
cells in patients with active vitiligo, indicating that this might
have a role in retarding the proliferation of surviving melanocytes
and recovery from vitiligo [17, 18, 38]. GM-CSF is a hematopoietic
growth factor. In vitro, it stimulates formation of
granulocyte-macrophage colonies and the proliferation of myeloid
progenitors. It has also been reported that it stimulates the
phagocytic function of mature granulocytes in vitro. As an indirect
function it might increase the activity of the granulocyte enzymes
(alkalene phosphatase, MPO, acid phosphatase) participating in
phagocytosis [30, 39]. Additionally, in an another study, it has
been reported that MPO is activated by GM-CSF [29].
A polymorphism located at the promotor region of MPO, is placed
at a distance of -463 base pairs of MPO gene. İt has been stated
that, by a MPO G-A base change, the mRNA expression and MPO level
were decreased, however, MPO production was increased by the G
allele in lung cancer with MPO G-463A [24]. It is known that a
genetically low level of MPO causes recurrent infections and lowers
the bactericidal activity of neutrophils [40]. In studies
investigating MPO gene polymorphism, different polymorphism types
have been reported [41]. In these studies, G-129 A
polymorphism, C-13/in1T and A-6/in11C polymorphism and G-463A
polymorphism were detected in beginning regions of
transcription.
In another study, it was stated that the A allele in
G-463 A polymorphism might be related to atherosclerosis risk
factors [41]. Wheatley et al. reported that decreased levels
of antioxidant activity might be associated with a high incidence
of the GG genotype of MPO G-463A polymorphism in the development of
pancreatic cancer, in a study investigating the relationship
between oxidative stress and pancreatic cancer [42]. In a study
investigating MPO gene polymorphism in patients with breast cancer,
it was reported that transcription of the A allele of the MPO
gene decreased enzyme levels [43]. It has been reported in
different studies that high levels of MPO expression in healthy
subjects and subjects with malignant myeloid series might be
related to different alleles of this enzyme [44]. Asselbergs
et al. stated that MPO-463 GA and AA genotypes decreased MPO
expression 2 fold when compared with the GG genotype [45]. In a
study investigating the association of MPO G-463A polymorphism with
infections in patients with multiple myeloma, the authors concluded
that there was no association [46]. To our knowledge, genes
responsible for the vitiligo have not yet been identified [1].
In the present study, MPO enzyme activity was significantly
lower in patients with vitiligo when compared with healthy
controls. There was no difference in terms of GG, GA and AA
genotype variance and allele distribution of MPO G-463A
polymorphism between healthy controls and subjects with vitiligo.
Lower levels of MPO enzyme activity might be associated with levels
of GM-CSF in subjects with vitiligo. On the contrary, it is not yet
known whether lower levels of MPO enzyme activity is associated
with polymorphism of MPO other than G-463A. However, the
interactions between levels of GM-CSF, IL-6 and MPO have not been
determined, which is a limitation of the present study.
In conclusion, the present study is the first study
investigating MPO G-463A polymorphism and enzyme levels, which
warrants further studies with higher patient numbers and a broader
polymorphism panel.
Acknowledgements
This work was supported by the Research Fund of Gaziantep
University (project numbers: TF.07.015). Conflict of interest:
none.
References
1 Castanet J, Ortonne JP. Pathophysiology of vitiligo.
Clin Dermatol 1997; 15: 845-51.
2 Ines D, Sonia B, Riadh MB, et al. A
comparative study of oxidant-antioxidant status in stable and
active vitiligo patients. Arch Dermatol Res 2006; 298: 147-52.
3 Dell’anna ML, Ottaviani M, Albanesi V,
et al. Membrane Lipid Alterations as a Possible Basis for
Melanocyte Degeneration in Vitiligo. J Invest Dermatol 2007; 18:
131-5.
4 Picardo M, Passi S, Morrone A, et al.
Antioxidant status in the blood of patients with active vitiligo.
Pigment Cell Res 1994; 7: 110-5.
5 Yildirim M, Baysal V, Inaloz HS, et al.
The role of oxidants and antioxidants in generalized vitiligo at
the tissue level. J Eur Acad Dermatol Venereol 2004; 18: 683-6.
6 Dell’anna ML, Maresca V, Briganti S,
et al. Mitochondrial impairment in peripheral blood
mononuclear cells during the active phase of vitiligo. J Invest
Dermatol 2001; 117: 908-13.
7 Jimbow K, Chen H, Park S, et al. Increased
sensitivity of melanocytes to oxidative stress and abnormal
expression of trosinase-related protein in vitiligo. Br J Dermatol
2001; 144: 55-65.
8 Koca R, Armutcu F, Altinyazar HC, et al.
Oxidant-antioxidant enzymes and lipid peroxidation in generalized
vitiligo. Clin Exp Dermatol 2004; 29: 406-9.
9 Agrawal D, Shajil EM, Marfatia YS, et al.
Study on the antioxidant status of vitiligo patients of different
age groups in Baroda. Pigment Cell Res 2004; 17: 289-94.
10 Dell’anna ML, Urbanelli S, Mastrofrancesco A,
et al. Alterations of mitochondria in peripheral blood
mononuclear cells of vitiligo patients. Pigment Cell Res 2003; 16:
553-9.
11 Yildirim M, Baysal V, Inaloz HS, et al.
The role of oxidants and antioxidants in generalized vitiligo. J
Dermatol 2003; 30: 104-8.
12 Passi S, Grandinetti M, Maggio F, et al.
Epidermal oxidative stress in vitiligo. Pigment Cell Res 1998; 11:
81-5.
13 Rokos H, Beazley WD, Schallreuter KU.
Oxidative stress in vitiligo: photo-oxidation of pterins produces
H2O2 and pteridin-6-carboxylic acid. Biochem Biophys Res Commun
2002; 292: 805-11.
14 Spencer JD, Gibbons NC, Rokos H, et al.
Oxidative stress via hydrogen peroxide affects proopiomelanocortin
peptides directly in the epidermis of patients with vitiligo. J
Invest Dermatol 2007; 127: 411-20.
15 Schallreuter KU, Moore J, Wood JM, et al.
In vivo and in vitro evidence for hydrogen peroxide (H2O2)
accumulation in the epidermis of patients with vitiligo and its
successful removal by a UVB-activated pseudocatalase. J Invest
Dermatol 1999; 4: 91-6.
16 Malle E, Furtmüller PG, Sattler W, et al.
Myeloperoxidase: a target for new drug development? Brit J
Pharmacol 2007; 152: 838-54.
17 Kemp EH, Waterman EA, Weetman AP. Immunological
pathomechanisms in vitiligo. Exp Rev Mol Med. 23 July, 2001;
http://www-ermm.cbcu.cam.ac.uk/01003362h.htm
18 Yu HS, Chang KL, Yu CL, et al.
Alterations in IL-6, IL-8, GM-CSF, TNF-alpha, and IFN-gamma release
by peripheral mononuclear cells in patients with active vitiligo. J
Invest Dermatol 1997; 108: 527-9.
19 Fenton JS, Bergstrom KG. Vitiligo: nonsurgical
treatment options and the evidence behind their use. J Drugs
Dermatol 2008; 7: 705-11.
20 Barrette WC, Hannum DM, Wheeler WD,
Hurst JK. General mechanism for the bacterial toxicity of
hypochlorous acid: Abolition of ATP production. Biochemistry 1989;
28: 9172-8.
21 Marcinkievicz J, Chain B, Nowak B,
Grabowska A, Bryniarski K, Baran J. Antimicrobial
and cytotoxic activity of hypochlorous acid: interactions with
taurine and nitrite. Inflamm Res 2000; 49: 280-9.
22 Candeias LP, Patella KB, Stratford MRL,
et al. Free Hydroxyl radicals are formed on reaction between
the neutrophil derived species superoxide anion and hypochlorous
acid. FEBS Lett 1993; 333: 151-3.
23 Cochrane CG. Cellular injury by oxidants. Am J Intern
Med 1991; 91: 23-30.
24 Taioli E, Benhamou S, Bouchardy C, et al.
Myeloperoxidase G-463 A polymorphism and lung cancer:
A HuGE genetic susceptibility to environmental carcinogens
pooled analysis. Genet Med 2007; 9: 67-73.
25 Hansson M, Olsson I, Nauseef WM. Biosynthesis,
processing, and sorting of human myeloperoxidase. Arch Biochem
Biophys 2006; 445: 214-24.
26 Gullberg U, Bengtsson N, Bulow E, et al.
Processing and targeting of granule proteins in human neutrophils.
J Immunol Methods 1999; 232: 201-10.
27 Bjerregaard MD, Jurlander J, Klausen P,
et al. The in vivo profile of transcription factors during
neutrophil differentiation in human bone marrow. Blood 2003; 101:
4322-32.
28 Deby-Dupont G, Deby C, Lamy M. Neutrophil
myeloperoxidase revisited: it’s role in health and disease.
İntensivmed 1999; 36: 500-13.
29 Matthias Z, Thomas CC, Claudia G, et al.
Evidence for the activation of myeloperoxidase by f-Meth-Leu-Phe
prior to its release from neutrophil granulocytes. Biochem Biophys
Res Commun 1997; 232: 209-12.
30 Kapp A, Kapp GZ, Danner M, et al. Human
granulocyte-macrophage colony stimulating factor: an effective
direct activator of human polymorphonuclear neutrophilic
granulocytes. J Invest Dermatol 1988; 91: 49-55.
31 Cascorbi I, Henning S, Brockmöller J,
et al. Substantially reduced risk of cancer of the
aerodigestive tract in subjects with variant--463A of the
myeloperoxidase gene. Cancer Res 2000; 60: 644-9.
32 Mason CP, Gawkrodger DJ. Vitiligo presentation in
adults. Clin Exp Dermatol 2005; 30: 344-5.
33 Schallreuter KU, Elwary SMA, Gibbons NCJ,
et al. Activation/deactivation of acetylcho- linesterase by
H2O2 more evidence for oxidative stres in
vitiligo. Biochem Biophy Res commun 2004; 315: 502-8.
34 Maresca V, Roccella M, Roccella F, et al.
Increased sensitivity to peroxidative agents as a possible
pathogenetic factor of melanocyte damage in vitiligo. J Invest
Dermatol 1997; 109: 310-3.
35 Arican O, Kurutas EB. Oxidative stress in the blood
of patients with active localized vitiligo. Acta Dermatovenerol Alp
Panonica Adriat 2008; 17: 12-6.
36 Kirnbauer R, Charvat B, Schauer E, et al.
Modulation of intercellular adhesion molecule-1 expression on human
melanocytes and melanoma cells: evidence for a regulatory role of
IL-6, IL-7, TNF beta, and UVB light. J Invest Dermatol 1992; 98:
320-6.
37 Imokawa G, Yada Y, Kimura M, et al.
Granulocyte/macrophage colony-stimulating factor is an intrinsic
keratinocyte-derived growth factor for human melanocytes in
UVA-induced melanosis. Biochem J 1996; 313: 625-31.
38 Tu CX, Gu JS, Lin XR. Increased interleukin-6
and granulocyte-macrophage clony stimulating factor levels in the
sera of patients with non-segmental vitiligo. J Dermatol Sci 2003;
31: 73-8.
39 Czygier M, Ławicki S, Szmitkowski M,
et al. Plasma granulocyte-macrophage colony stimulating factor
(GM-CSF) and activity of enzymes in granulocytes of breast cancer
patients. Przegl Lek 2006; 63: 654-7.
40 Kitahara M, Eyre HJ, Simonian Y, et al.
Hereditary myeloperoxidase deficiency. Blood 1981; 57: 888-93.
41 Hoy A, Trégouët D, Leininger-Muller B,
et al. Serum myeloperoxidase concentration in a healthy
population: biological variations, familial resemblance and new
genetic polymorphisms. Eur J Hum Genet 2001; 9: 780-6.
42 Paul Wheatley-Price, Asomaning K, Reid A, et al.
Myeloperoxidase and Superoxide Dismutase Polymorphisms Are
Associated With an Increased Risk of Developing Pancreatic
Adenocarcinoma. Cancer 2008; 112: 1037-42.
43 Piedrafita FJ, Molander RB, Vansant G,
et al. An Alu element in the myeloperoxidase promoter contains
a composite SP1-thyroid hormone-retinoic acid response element. J
Biol Chem 1996; 271: 14412-20.
44 Reynolds W, Chang E, Douer D. An allelic
association implicates myeloperoxidase in the etiology of acute
promyelocytic leukemia. Blood 1997; 90: 2730-7.
45 Asselbergs FW, Reynolds WF, Cohen-Tervaert JW,
et al. Myeloperoxidase polymorphism related to cardiovascular
events in coronary artery disease. Am J Med 2004; 116: 429-30.
46 Molle I, Ostergaard M, Melsvik D, et al.
Infectious complications after chemotherapy and stem cell
transplantation in multiple myeloma: Implications of Fc gamma
receptor and myeloperoxidase promoter polymorphisms. Leukemia &
Lymphoma 2008; 49: 1116-22.
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