ORIGINAL PAPER

The relationship between severity of the disease and circulatingnucleosomes in psoriasis patients

Aziz Ramazan Dilek • Nursel Dilek •

Yunus Saral • Derya Yuksel

Received: 4 December 2012 / Revised: 20 March 2013 / Accepted: 22 March 2013

� Springer-Verlag Berlin Heidelberg 2013

Abstract Psoriasis was initially considered to represent a

disease of abnormal epidermal keratinocyte proliferation.

Proliferation of keratinocytes is restricted by apoptotic cell

death to maintain a constant thickness of epidermis.

Nucleosomes are mainly released by apoptotic cells. Tumor

necrosis factor-a (TNF-a) is an important factor affecting

the apoptosis. In the present study, the relationship between

TNF-a, nucleosome and the Psoriasis Area and Severity

Index (PASI) score was investigated. The patients were

divided according to PASI score into three groups (mild,

moderately, severe). Serum TNF-a and nucleosome levels

were measured using Enzyme-linked immunosorbent assay

(ELISA) method. Our findings show a correct relationship

between PASI scores and TNF-a and an inverse relationship

between nucleosome and PASI score. According to the

results obtained from the study, we believe that serum

nucleosome levels can be used as a new indicator in follow-

up of patients with psoriasis and monitoring of the effec-

tiveness of drugs which used in the treatment of psoriasis.

Keywords Psoriasis � Nucleosome � TNF-a

The relationship between severity of the disease

and circulating nucleosomes in psoriasis patients

Psoriasis was initially considered to represent a disease of

abnormal epidermal keratinocyte proliferation, manifesting

in the characteristic thickening and scaling of erythematous

psoriatic plaques [3]. Proliferation of keratinocytes is

restricted by apoptotic cell death to maintain a constant

thickness of epidermis [5]. Upregulation of cell survival

pathways and suppression of apoptosis are implicated in

skin inflammation and maintenance of epidermal hyper-

plasia in psoriasis [11]. Suppression of apoptosis of T

lymphocytes leads to their survival which relates to the

chronic and relapsing characters of psoriasis [6]. An

important cytokine that is associated with keratinocyte

proliferation in psoriasis is (TNF-a) [7]. TNF-a is a mul-

tifunctional cytokine that mediates inflammation, immune

responses and apoptosis [10]. Apoptosis is a complex,

highly regulated and active physiologic process by which

organisms regulate their cell growth and tissue remodeling

in such a manner that will neither injure neighboring cells

nor elicit any inflammatory reaction [14]. Nucleosome core

particles are protein complexes highly conserved in all

eukaryotes and primarily responsible for compaction of the

eukaryotic genome and also play a role in various cellular

processes, including transcriptional gene regulation, DNA

replication, and DNA repair [8]. Elevated concentrations of

circulating nucleosomes were also observed in other

pathological situations, such as trauma, sepsis, stroke and

autoimmune diseases [13]. Unfortunately, there is no

research in the literature about the relationship in men-

tioned biomarkers in etiopathogenesis of psoriasis. In the

present study, we aimed to investigate the relationship

between TNF-a, nucleosome and the PASI score.

Methods

A total of 78 psoriasis patients (36 females, 42 males, mean

age 36.60 ± 7.8 years) consulting the Department of

A. R. Dilek (&)

Microbiology Department of Recep Tayyip Erdogan University,

Medical Faculty Hospital, Rize 53000, Turkey

e-mail: ar.dilek@hotmail.com

N. Dilek � Y. Saral � D. Yuksel

Dermatology Department of Recep Tayyip Erdogan University,

Medical Faculty Hospital, Rize, Turkey

123

Arch Dermatol Res

DOI 10.1007/s00403-013-1347-4

Dermatology, during September 2011 and February 2012

were included in the study. The patients who have additional

disease such as infection, cancer, stroke and autoimmune

diseases were excluded from the study. In addition, the

patients who receive systemic therapy recently (the last

15 days) were excluded from the study. The patients were

divided according to PASI score into three groups: group 1

with PASI score up to 2 (28 patients), group 2 with PASI

score more than 2 up to 10 (25 patients) and group 3 with

PASI score more than 10 (25 patients). After blood collection

for TNF-a measurement, serum of all patients was imme-

diately obtained by centrifugation, transferred into cryotu-

bes, and stored at -70 �C until assayed. The serum samples

for nucleosome determination were centrifuged at 3,000g for

15 min and treated with 10 mmol/l EDTA (ethylenediami-

netetraacetic acid) immediately after centrifugation and

stored at -70 �C until further analysis. Serum TNF-a was

measured using ELISA method (Human TNF-a ELISA kit,

Anogen, Canada) according to the manufacturer’s protocol.

Absorbance (OD) of each well determined at 450 nm with a

microtiter plate reader (Multiskan GO, Thermo Scientific,

Waltham, MA, USA) in the 5th minute. Standard curves

were fitted using Titri ELISA software. The fitted curve was

then used to convert sample absorbance readings to TNF-aconcentration. The commercially available Cell Death

Detection ELISAPLUS Kit (Roche Diagnostics Mannheim,

Germany) was used according to the manufacturer’s

instructions for quantification of nucleosome concentrations

in serum. In this kit, walls of microplate are coated with

streptavidin. Serum samples and two monoclonal mouse

antibodies, which are directed against histones and DNA,

were added to the plate and incubated, and this allows the

specific determination of mono- and oligonucleosomes. In

the incubation period, anti-histone antibody binds to the

histone-component of the nucleosomes and simultaneously

captures the immunocomplex to the streptavidin-coated

microplate via its biotinylation. Additionally, the anti-DNA-

POD antibody reacts with the DNA-component of the

nucleosomes. After removal of the unbound components, the

anti-DNA antibody, which is labeled with peroxidase, reacts

with the 2,20-azino-di-(3-ethylbenzthiazoline-sulfonate)

substrate. The resulting color development is proportional to

the amount of nucleosomes which are captured in the anti-

body sandwich and enables the photometric quantification of

nucleosomes. Nucleosome concentrations were calculated

using a standard curve in ng/mL.

Statistics

All statistical analyses were performed using SPSS software

for Windows (Version 18.0). One-sample Kolmogorov–

Smirnov test was used to show the normal distribution of

the test parameters. Homogeneity of variances was ana-

lyzed using Levene’s test and ANOVA test was used for

comparing between the group variance. Post hoc tests were

performed (Bonferroni) for examining the relationship

between the groups. In addition, a non-parametric method,

the Kruskal–Wallis test, was performed for comparing.

Results

Distribution of the test parameters (TNF-a, nucleosome)

was normal for each test (one-sample Kolmogorov–Smir-

nov test, Figs. 1, 2). Homogeneity of variances was found

to be homogeneous. ANOVA test results are displayed in

Tables 1 and 2. Significant difference was detected

between the groups (P \ 0.05) in a non-parametric test

(Kruskal–Wallis). Our findings show a direct relationship

between PASI scores and TNF-a and an inverse relation-

ship between nucleosome and PASI score (Figs. 3, 4).

While a significant increase for TNF-a with higher PASI

score is noteworthy, a decrease in serum nucleosome levels

was seen with increasing PASI score (Tables 3, 4).

Discussion

Cell death is an important molecular event during cell life,

since unwanted cells can be removed through apoptosis.

Multiple types of cell death have been found, including

apoptosis, autophagy, necrosis, senescence, and mitotic

Fig. 1 Distribution of the serum TNF- a levels

Arch Dermatol Res

123

catastrophe [15]. In chronic inflammatory diseases, such as

psoriasis, rheumatoid arthritis or crohn disease, the rate of

apoptosis is reduced in the inflamed tissue, and defective

apoptosis may be a causative factor for inflammation. In

psoriasis, it is thought that hyperproliferation of keratino-

cytes, in concert with their resistance to apoptosis, induces

epidermal acanthosis [11]. Programmed necrosis is depen-

dent on a death receptor-mediated signal transduction

pathway, such as tumor necrosis factor (TNF) receptor-

mediated cell death pathway [15]. TNF-a is produced by

both activated dendritic cells and T cells, including T helper

1 (Th1), Th17 and Th22 [1]. TNF acts through two distinct

receptors; TNFR1 is constitutively expressed on virtually all

nucleated cell types, TNFR2 is generally inducible and is

preferentially expressed on endothelial and hematopoietic

cells. Engagement of TNFRs initiates a cascade of events

leading to three main types of immune regulation. The first is

the nuclear factor jB (NF-jB) activation and the promotion

of inflammation. The second involves mitogen-activated

protein kinase (MAPK) and c-Jun N-terminal kinase; the

latter promotes cellular differentiation, proliferation and

apoptosis. The third is associated with death signaling [12].

TNF-a concentrations are higher in psoriatic lesions than in

unaffected skin of psoriatic patients and tend to decline with

clearing of the lesions after effective therapy [7].

Fig. 4 Error bar represent 95 % confidence interval of nucleosome

Fig. 2 Distribution of the serum nucleosome levels

Table 1 Comparing between the group variance for TNF-a(ANOVA test)

Sum of

squares

df Mean

square

F Sig.

Between

groups

5,581.915 2 2,790.958 68.543 0.000

Within groups 3,053.898 75 40.719

Total 8,635.813 77

Table 2 Comparing between the group variance for nucleosome

(ANOVA test)

Sum of

squares

df Mean

square

F Sig.

Between

groups

676.550 2 338.275 185.760 0.000

Within groups 136.577 75 1.821

Total 813.127 77

Fig. 3 Error bar represent 95 % confidence interval of TNF- a

Arch Dermatol Res

123

Small amounts of nucleosomes, found in the plasma and

serum of healthy persons, are believed to be released

during or after physiological cell death. In addition, lym-

phocytes may secrete nucleosome directly, or they may be

liberated from cells, e.g., erythroblasts, during differentia-

tion. [9]. Nucleosomes are mainly released by apoptotic

cell death. Under physiological conditions, the nucleo-

somes are packed into membrane-bound vesicles and

engulfed by macrophages and neighboring cells. In the case

of a high rate of apoptosis, these phagocytosing mecha-

nisms are saturated or impaired, leading to higher con-

centrations of nucleosomes in the circulation [2]. The

concentrations of circulating cell-free DNA (cf-DNA)

increase in various benign and malignant pathologic con-

ditions, including cancers. In serum and plasma, DNA is

thought to exist predominantly as mononucleosomes and

oligonucleosomes [4]. Several studies were conducted

examining the situation of this apoptotic cell death marker

in a variety of clinical situations [2, 9]. In a more recent

study, the effectiveness of chemotherapeutics was investi-

gated by measuring the concentration of serum nucleo-

somes and successful results have been obtained [9]. In

another study, diagnostic/prognostic value of circulating

nucleosomes in sepsis was investigated and similarly suc-

cessful results were obtained [2].

However, the diagnostic/prognostic value of circulating

nucleosomes in psoriasis patients remains unknown. In our

study, we examined the amount of circulating TNF-awhich play an important role in apoptosis and nucleosome

and our findings showed that, there was a correct rela-

tionship between PASI scores and TNF-a and an inverse

relationship between nucleosome and PASI score. These

striking results suggests that serum nucleosomes levels can

be used as a new marker in follow-up of patients with

psoriasis and monitoring of the effectiveness of drugs

which used in the treatment of psoriasis. However, this is

the first report; therefore, larger study groups are needed to

validate the appropriateness of these findings.

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