International Immunology, Vol. 21, No. 3, pp.

237243
doi:10.1093/intimm/dxn142

The Japanese Society for Immunology. 2009. All rights reserved.

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Serum DNase I, soluble Fas/FasL levels and cell

surface Fas expression in patients with SLE:
a possible explanation for the lack of efficacy of
hrDNase I treatment
Elisa Tinazzi1,*, Antonio Puccetti2,3,*, Roberto Gerli4, Antonella Rigo5, Paola Migliorini6,
Sara Simeoni1, Ruggero Beri1, Marzia Dolcino2, Nicola Martinelli1, Roberto Corrocher1 and
Claudio Lunardi1
1

Department of Clinical and Experimental Medicine, Section of Internal Medicine, University of Verona, 37134 Verona, Italy
Institute G. Gaslini, Department of Immunology, Genova, Italy
3
Department of Experimental Medicine, Section of Histology, University of Genova, Genova, Italy
4
Department of Clinical and Experimental Medicine, Section of Rheumatology, University of Perugia, Perugia, Italy
5
Department of Clinical and Experimental Medicine, Section of Hematology, University of Verona, Verona, Italy
6
Department of Internal Medicine, Section of Clinical Immunology, University of Pisa, Pisa, Italy
2

Abstract
The objectives of the study are to evaluate DNase I serum levels and their correlation with soluble Fas
(sFas) and soluble Fas ligand (sFasL) and with cell surface Fas expression in patients with systemic
lupus erythematosus (SLE), thus contributing to the dysregulated apoptosis typical of the disease.
The methods include the following: Serum DNase I levels in patients and in controls were detected
using the dot blot method and quantified by densitometry; sFas and sFasL were quantified using an
ELISA system. Cell surface Fas expression was evaluated by FACS analysis. Apoptosis was studied
by means of internucleosomal DNA degradation using a commercially available kit. The results are as
follows: We found a significant difference in DNase I, sFas and sFasL serum levels between patients
and controls. Levels of DNase I <7.79 ng ml21 are more represented in patients with SLE. Active SLE is
strongly associated with high sFas levels and detectable sFasL. DNase I does not correlate with sFas
or sFasL, whereas it correlates with T cell surface Fas expression that is higher in patients with active
SLE than in healthy controls. Finally, administration of exogenous human recombinant DNase
(hrDNase) I to freshly isolated T cells up-regulates cell surface Fas expression and induces increased
susceptibility to Fas-mediated apoptosis. In conclusion, our findings confirm that DNase I is low in
SLE and suggest that it may play a role in apoptosis in SLE by regulating the surface expression of
the cell death molecule Fas. This role may contribute to explain the inefficacy of hrDNase I in SLE,
a treatment proposed for the ability of DNase I to remove DNA from auto-antigenic nucleoprotein
complexes.
Introduction
Systemic lupus erythematosus (SLE) is characterized by the
presence of anti-nuclear antibodies (ANAs) directed against
naked DNA and nucleosomes. The etiology of SLE is unknown, but several studies suggest that increased production and/or inadequate clearance of nuclear DNAprotein
complexes after cell death may initiate and/or propagate the
disease (1).

DNases are the primary enzymes involved in the metabolism

and clearance of DNA. Low serum and urine DNase activity has
been observed in some patients with SLE (24), as well as in
lupus-prone mice (5); indeed DNase I-deficient mice have been
shown to develop a lupus-like phenotype, with nephritis and
serum auto-antibodies directed against nuclear antigens (6).
Reduction or loss of DNase I activity, such as in case of gene

*These authors contributed equally to this study.

Correspondence to: C. Lunardi; E-mail: claudio.lunardi@univr.it
Transmitting editor: L. Moretta

Received 8 September 2008, accepted 8 December 2008

Advance Access publication 30 January 2009

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Keywords: DNase I, human recombinant DNase I, SLE, soluble Fas/FasL

238 DNase I and SLE

IgG antibody (Sigma, Milan, Italy). DNase I level in serum
samples was calculated comparing the density of each dot
with dots containing known concentrations of hrDNase I
(purchased from HoffmanLa Roche, Grenzach-Wyhlen,
Germany) using a VersaDoc image analyzer apparatus (BioRad, Hercules, CA, USA).
Measurement of human sFas and sFasL
Serum levels of sFas and sFasL were quantified using commercially available ELISA kits (Bender MedSystems, Wien,
Austria) following the manufacturers instructions. The detection limit was of 20 pg ml 1 and 0.1 ng ml 1 for sFas and
sFasL, respectively.
Cells preparation and culture
Peripheral blood mononuclear cells (PBMC) were isolated
from freshly heparinized PB of 18 patients and 10 controls
by density-gradient centrifugation on Lymphoprep (Nycomed
Pharma, Oslo, Norway) and analyzed by flow cytometry.
In four patients and two controls, PBMC were suspended
at 2 3 106 ml 1 in RPMI-1640 + 10% FCS incubated with 80
ng ml 1 of hrDNase I for 96 h at 37
C. Then, they were analyzed by flow cytometry.
Flow cytometry analysis
FITC-conjugated anti-CD3 (anti-CD3FITC), R-PE-conjugated
anti-CD95/Fas (anti-CD95PE) mAb or isotype/fluorochromematched controls were purchased from Becton Dickinson
(San Jose, CA, USA). Data were collected on a FACSCalibur
(Becton Dickinson) using CellQuest software and analyzed
by FlowJo software (TreeStar).
Measurement of apoptosis

Seventy-three SLE patients fulfilling the revised criteria for SLE of

the American College of Rheumatology (23) and 60 age- and
sex-matched healthy controls were enrolled. Twenty-two patients
were in remission, 29 had moderate active and 22 had active
SLE according to the SLE disease activity index (24).
Blood samples were drawn from patients and controls after informed oral or written consent.

The extent of internucleosomal DNA fragmentation was

quantified using a commercially available kit (Roche Biochemical, Indianapolis, IN, USA) according to the manufacturers instructions. The principle of this test is based on the
detection of mono- and oligonucleosomes in the cytoplasmic
fractions of cell lysates. The enrichment of mono- and oligonucleosomes released into the cytoplasm is calculated by
dividing the absorbance value obtained in the cells exposed
to the apoptotic stimuli with the absorbance value of the untreated cells (absorbance of sample cells/absorbance of
control cells). The enrichment factor was used as an index
of apoptosis (apoptotic index) and is shown on the vertical
axis as mean 6 SD of triplicates.
For the induction of apoptosis, cells were incubated with
either the anti-Fas IgG3 mAb (Alexis, Launsen, Switzerland)
or the recombinant human sFasL (Alexis) and cell death
assessed at several time points. To inhibit Fas-mediated apoptosis, cells were incubated with the blocking anti-Fas
IgG2b mAb (Alexis).

Serum levels of DNase I

Statistical analysis

Patients and controls sera were blotted onto a nitrocellulose

membrane (Hybond-C Extra, Amersham Biosciences, Freiburg, Germany), incubated overnight with a rabbit antiDNase I antibody developed in our laboratory (11) and then
revealed with a peroxidase-conjugated mouse anti-rabbit

Calculations were performed with SPSS 14 statistical package. Quantitative data with a normal distribution were
expressed as mean 6 SD and were analyzed with Students
t-test. DNase I and sFas, which presented a not-normal distribution, were expressed as median with 2575
percentile

Materials and methods

Patients

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mutation of DNase I (7), may result in a high risk to produce

ANAs as a potential prerequisite to develop a SLE-like disease.
Besides the removal of DNA from auto-antigenic nucleoprotein complexes to prevent immune stimulation, DNase I plays
a role also in apoptosis (810). We have previously demonstrated that DNase I is responsible for internucleosomal DNA
degradation in human cells undergoing apoptosis in response to pharmacological stimuli (11) and that this endonuclease behaves as a transcription factor that regulates cell
surface Fas expression (12). Moreover, exogenous human
recombinant DNase (hrDNase) I is endocytosed into cells
up-regulating cell surface Fas expression (12).
Fas is an apoptosis signaling receptor belonging to the
tumor necrosis factor (TNF) receptor family, while FasL is a type
II membrane protein of the TNF family, which behaves as a death
factor, transducing death signals to Fas-positive cells. The hypothesis that perturbation in the Fas/FasL system may be responsible for the development of autoimmunity has prompted
studies in patients with autoimmune disorders leading to controversial results (1315). An abnormal regulation of the Fas/FasL
apoptotic signaling pathway has been associated with the
pathogenesis of SLE (16, 17). In particular, the extensive apoptosis of T lymphocytes (18), of bone marrow CD34+ cells
(19), of neutrophils (20) and of monocytes/maturing macrophages (21), as well as the resistance of autoimmune Th cells
driving pathogenic auto-antibody production (22), seem to result from impaired activation of Fas/FasL system.
In light of the results of our previous study showing a direct
link between DNase I and fas gene expression (12), we decided first to evaluate DNase I serum levels in SLE patients,
second to evaluate whether DNase I correlates with soluble
Fas (sFas) and soluble Fas ligand (sFasL) and third whether
DNase I correlates with cell surface Fas expression. Moreover, we wanted to evaluate whether these molecules may
be useful in predicting either the development of SLE or the
organ involvement or in discriminating different clinical
stages of the disease.
Finally, we wanted to confirm whether exogenous hrDNase I
is able to up-regulate cell surface Fas expression and facilitate
Fas-mediated apoptosis in freshly isolated peripheral blood
(PB) T cells obtained from SLE patients and from healthy subjects, as already shown in human cell lines (12).

DNase I and SLE 239

Results
Serum DNase I levels are low in SLE
The characteristics of the study population and the levels of
DNase I, sFas and sFasL are shown in Table 1. Serum
DNase I levels were lower in SLE compared with controls
(11.75 ng ml 1 in SLE patients versus 13.11 ng ml 1 in control subjects; P = 0.002 by MannWhitney test). Moreover,
considering low levels of DNase I (<7.79 ng ml 1: below the
arbitrary threshold value of the 20th percentile in the healthy
controls), subjects with low DNase I levels were significantly
more represented in SLE patients than in controls [42.4 versus 18.3%; P = 0.003 by Chi-square test; odds ratio (OR)

Table 1. General characteristics of the study population

Male/female
Age (years)
Disease activity
Quiescent
Mild active
Active
Renal involvement
ANA positive
Anti-nativeDNA+
DNase I (ng ml 1)a
Subjects with DNase
I level <7.79 ng ml 1
sFas (pg ml 1)a
Subjects with sFas
level <176.9 pg ml 1
Subjects with dosable
sFasL
a

Controls (n = 60)

SLE subjects (n = 73)

9/51 (19.1%)
38 6 10.9

7/66 (11%)
40.5 6 12.9

22/73
29/73
22/73
25/73
65/73
23/73
11.75
31/73

0
0
13.11 (8.9923.2)
11/60 (18.3%)

(30.1%)
(39.7%)
(30.1%)
(34.2%)
(89%)
(31.5%)
(3.6918.53)b
(42.4%)c

211.7 (183.7262.4) 134.7 (91276.6)b

11/60 (18.3%)
46/73 (63%)c
12/60 (20%)

49/73 (67.1%)c

Data are expressed as median with 2575
percentile range.

P < 0.05 by MannWhitney test.
P < 0.05 by Chi-square test.

b
c

3.34 with confidence interval (CI) 95% 1.407.49]. These

data indicate that serum DNase I is reduced in patients with
SLE when compared with healthy donors and that levels of
DNase I <7.79 ng ml 1 are associated with SLE.
sFas and sFas ligand levels in SLE
Also sFas levels were lower in SLE patients than in control
subjects (sFas: 134.7 versus 211.7 pg ml 1; P = 0.002 by
MannWhitney test) and subjects with low sFas levels
(<176.9 pg ml 1) were more represented in SLE patients
(63 versus 18.3% of controls; P < 0.001 by Chi-square test;
OR 7.22 with CI 95% 2.818.6).
Considering sFasL, 67.1% of patients and only 20% of
controls have a dosable sFasL (P < 0.001 by Chi-square
test; OR 8 with CI 95% 3.0720.88). Therefore, low sFas levels and dosable sFasL are typical features of SLE.
We then stratified the study population on the basis of low
sFas status and of dosable sFasL (Table 2). A highly significant asymmetry was observed (total Chi-square = 49.46,
P < 0.001; Chi-square for linear trend = 43.04, P < 0.001;
remaining Chi-square = 6.42, P = 0.04): remarkably, only
one of the 73 SLE patients presented high levels of sFas
and not dosable sFasL. On the other hand, only two of 60
control subjects had low levels of sFas and dosable sFasL.
If we consider the SLE diagnosis on the basis of the presence of either low sFas levels or dosable sFasL, we have
a test with high sensibility (98.1%) and low specificity
(67.5%). If we consider the SLE diagnosis on the basis of
the concomitant presence of both sFas low levels and dosable sFasL, we have a test with high specificity (97.5%) and
low sensibility (31.5%).
When low DNase I status, low sFas status, dosable sFasL
and also age and sex were simultaneously controlled in
a multiple logistic regression analysis, low sFas levels
(P < 0.001; OR 69.7 with CI 95% 9.2526.2) and dosable
sFasL (P = 0.001; OR 18.4 with CI 95% 3.1111.1) maintained
a significant association with SLE, whereas this association
was lost for low DNase I (P = 0.517; OR 1.76 with CI 95%
0.329.76). Moreover, there was a significant direct correlation
between sFas and sFasL (correlation coefficient 0.29;
P = 0.003 by Kendalls rank correlation), while there was no
correlation between sFas or sFasL and DNase I. These data
indicate that, among the three variables studied (sFas, sFasL
and DNase I), sFas and sFasL have a better ability than
DNase I in discriminating SLE patients from controls.
DNase I, sFas, sFasL and SLE disease activity
Next we evaluated whether the three variables studied
(DNase I, sFas and sFasL) correlate with SLE serological
and clinical parameters.
DNase I, sFas and sFasL levels did not correlate with either inflammation markers (ESR and CRP), presence or absence of anti-double strand DNA antibodies and of renal
involvement (data not shown).
SLE patients were divided into three groups on the basis
of disease activity (quiescent, mild active and active): the
levels of sFas and sFasL were significantly different in the
three patients subsets. On the contrary, no significant difference was found in DNase I levels (Table 3).

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intervals and were analyzed with non-parametric tests

(MannWhitney or KruskalWallis with all pairwise comparisons). Qualitative data were analyzed with the Chi-square
test or Fishers exact test when indicated. Because a high
rate of subjects did not present a detectable level of sFasL,
data are expressed as dichotomous variable (detectable or
not detectable). Low DNase I or sFas levels are defined as
those below the arbitrary threshold value of the 20th percentile in the control group.
A multiple logistic regression analysis that simultaneously
controlled DNase I, sFas, sFasL status and also sex and
age was performed to evaluate the independent association
between these variables and SLE. The study population was
then stratified into many groups on the basis of DNase, sFas
and sFasL status and the association of these subgroups
and SLE was analyzed.
DNase I, sFas and sFasL status were also analyzed in
SLE subjects divided on the basis of disease activity or renal
involvement.
Correlations between DNase I, sFas, sFasL, cell surface Fas
levels, erythrocyte sedimentation rate (ESR) and C reactive
protein (CRP) were analyzed with Kendalls tau test.
A value of P <0.05 was considered significant.

240 DNase I and SLE

Table 2. Distribution of sFas and sFasL status stratification subgroups in SLE and controls

Controls (n = 60)
SLE (n = 73)

High sFas and

not dosable sFasL

High sFas and

dosable sFasL

Low sFas and

not dosable sFasL

Low sFas and

dosable sFasL

40 (66.6%)
1 (1.4%)

11 (18.3%)
26 (35.6%)

7 (11.7%)
23 (31.5%)

2 (3.3%)
23 (31.5%)

Total Chi-square = 49.46; P < 0.001; Chi-square for linear trend = 43.04; P < 0.001; remaining Chi-square = 6.42; P = 0.04.

Remarkably, SLE patients with quiescent disease resulted to

have lower levels of serum sFas (108.5 pg ml 1; P < 0.05 by
KruskalWallis all pairwise comparisons) when compared not
only with control subjects (211.7 pg ml 1) but also with SLE
patients with active disease (286.1 pg ml 1). No significant
difference was found between controls and SLE patients with
active disease; however, the latter group presented a trend
toward higher sFas levels. Low levels of sFas (<176.9 pg
ml 1) was therefore mainly a characteristic of SLE patients
with quiescent disease (21/2295.5% versus 8/2236.4% in
SLE patients with active disease; P < 0.001 by Chi-square
test). On the other hand, the proportion of subjects with dosable sFasL increased significantly and progressively from
quiescent to active disease (18/2281.8% versus 8/22
36.4% in SLE patients with quiescent disease; P = 0.011 by
Chi-square test) (Fig. 1). These data show that the combined
utilization of sFas and sFasL levels allows a better discrimination among quiescent, mild active and active SLE.
DNase I levels, cell surface Fas expression and apoptosis
Since we have previously shown that over-expressed or exogenously administered DNase I up-regulates Fas expression in different cell lines, we analyzed the cell surface
expression of Fas (Fig. 2A) in a group of 28 subjects (18
patients with active SLE and 10 controls). There was a mild

Discussion
Clearance of nucleosomes from the body is crucial for the development of SLE; DNase I is responsible for nucleosome degradation and therefore low enzyme activity may contribute to
the anti-nuclear auto-antibody production typical of SLE (25,
26). The decreased activity may be related to mutation in the
DNase I gene as shown in 2 of 200 Japanese patients studied (7). However, this mutation is not present in the three major ethnic groups: Caucasian, African and Asian (27). Singlenucleotide polymorphism (SNP) may affect or not the serum
DNase I activity (28, 29) and from this effect may depend
the described association of a SNP with colorectal (30) and
gastric carcinomas (31) and with myocardial infarction (32).
The measurement of serum DNase I activity may be influenced by a number of factors such as (i) the presence of
natural inhibitors of the enzyme (actin), (ii) the presence of
anti-DNA antibodies able to bind the catalytic site of DNase
I and to interfere with DNase I activity (33) and (iii) the

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Fig. 1. Distribution of subjects with low sFas levels (<176.9 pg ml 1;

gray bars) and dosable sFasL (white bars) in controls and in SLE
patients divided on the basis of disease activity. *: Significant
difference from control group; P < 0.05 by Chi-square test. #:
Significant difference from SLE patients with active disease; P < 0.05
by Chi-square test. : Significant difference from SLE patients with
quiescent disease; P < 0.05 by Chi-square test.

significant direct correlation between DNase I level and Faspositive-CD3+ lymphocytes (correlation coefficient 0.275;
P = 0.045 by Kendalls rank correlation). Interestingly, the percentage of Fas-positive-CD3+ lymphocytes was significantly
higher in SLE patients than in controls (P < 0.05) (Fig. 2B).
Finally, we wanted to verify whether exogenous hrDNase I
can modulate Fas expression in freshly isolated cells. For
this purpose, PBMC obtained from four patients with SLE
and two healthy donors were incubated with hrDNase I and
stained with anti-Fas mAb. Indeed, an increased expression
of Fas was detectable on CD3+ cells by FACS analysis after
incubation with hrDNase I in cells obtained from both SLE
patients and healthy donors (Fig. 3A). These data show that
exogeneously administered hrDNase I is able to modulate
Fas expression also in freshly isolated cells.
Since Fas/FasL-mediated pathway is particularly important in
T cells undergoing apoptosis in SLE (18), we evaluated
whether cells exposed to hrDNase I had an increased susceptibility to Fas-mediated apoptosis. We observed that
mononuclear cells, obtained from the same subjects analyzed
for cell surface Fas induction and incubated with hrDNase I
as above described, showed an increased internucleosomal
DNA degradation upon engagement of Fas using either an
anti-Fas IgG3 mAb or recombinant FasL (Fig. 3B and C).
The apoptosis observed was truly Fas dependent since
this phenomenon was not observed in the presence of an
anti-Fas-blocking antibody (data not shown). These results
show that exogenously administered hrDnase I can up-regulate
Fas expression and induce Fas-mediated apoptosis also in
freshly isolated cells derived from both normal donors and
SLE patients.

DNase I and SLE 241

Table 3. DNase I, sFas and sFasL levels in SLE patients divided on the basis of disease activity
Controls (n = 60)

1 a

DNase I (ng ml )
Subjects with DNase I <7.79 ng ml
sFas (pg ml 1)a,b
Subjects with sFas<176.9 pg ml 1d
Subjects with dosable sFasLd

13.11
9/47
211.7
12/60
12/60

(8.9923.2)
(19.1%)
(183.7262.4)
(20%)
(20%)

SLE (n = 73)
Quiescent (n = 22)

Mild active (n = 29)

Active (n = 22)

12.3
7/22
108.5
21/22
8/22

7.5
15/29
154.3
18/29
22/29

12.1
7/22
286.1
8/22
18/22

(6.618.5)
(31.8%)
(78.6129.8)c
(95.5%)e
(36.4%)

(2.918.3)
(51.7%)
(88.9251.4)
(62%)e
(75.9%)e

(3.518.1)
(31.8%)
(120.8317.1)
(36.4%)
(81.8%)e

Data are expressed as median with 25th75th percentile range.

P < 0.001 by KruskalWallis.
Significant difference from controls (P < 0.001) and from SLE patients with active disease (P = 0.03) by KruskalWallis all pairwise comparisons
(DwassSteelChritchlowFligner).
d
P < 0.001 Chi-square test.
e
Significant difference from control group; P < 0.05 by Chi-square test.
b
c

measurement of other endonucleases activity, using commercially available kits. Because of these problems and because
of our previous findings on the ability of exogenous DNase I
molecule to enter into living cells by endocytosis and to modulate surface Fas expression, we decided to measure the DNase I protein level. We found that DNase I molecule is
decreased in SLE patients, as already reported for the enzyme
activity (24). Indeed, a DNase I level <7.79 ng ml 1 is more
represented in SLE patients, with an OR of above three
times. However, DNase I levels did not correlate with organ
involvement or disease activity, as already reported (4), and
with sFas and sFasL. Increased levels of sFas and detectable sFasL were typical of active SLE, in line with the aberrant levels of apoptosis present in the active phase of the
disease. Indeed, our results are in line with previous reports
describing that in active lupus serum levels of both sFas
and sFasL are increased compared with those of patients
with inactive disease and correlate with over-expression of
cell surface Fas by T cells and with T cell apoptosis (34, 35).
Serum DNase I correlates with surface expression of Fas
in PB T lymphocytes obtained from patients with active SLE.

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Fig. 2. Surface expression of CD95/Fas on T lymphocytes from SLE

and normal subjects. Panel (A): PBMC were labeled with anti-CD3
FITC and anti-CD95PE mAbs. Representative histograms of three
SLE patients (red) and three controls (blue) are shown. Samples were
analyzed by FlowJo software (TreeStar). Panel (B): percentage of
CD3+ Fas+ cells in peripheral blood of 18 SLE patients and (red
circles) and 10 healthy controls (blue circles). The mean values 6
SDs were 36 6 19 and 24 6 10, respectively (P = 0.046).

Most importantly, we confirmed the ability of hrDNase I to enter

into freshly isolated T cells and to increase surface Fas expression in PB T cells as already shown in the erythroleukemia cell
line K562 (12). A possible explanation for the direct correlation between DNase I levels and cell surface Fas expression
in SLE patients is that circulant DNase I enters into the cells
through the binding to the cation-independent mannose 6phosphate/insulin-like growth factor (CI-MPR) receptor and
induces surface expression of the death receptor Fas, behaving as a transcription factor for the fas gene. In this way, DNase I would contribute to the pathogenesis of SLE not only
through removal of DNA from auto-antigenic nucleoprotein
complexes but also by modulating Fas expression and ultimately cell apoptosis. Indeed, the results obtained incubating
PBMC from patients and controls with hrDNase I on the expression of Fas and on increased susceptibility to apoptosis
suggest that such a mechanism may be present also in vivo.
hrDNase I has been proposed as a therapeutic agent in
SLE (26, 36); however, no changes both in clinical manifestations and in serum markers of disease activity have
been observed in humans. Moreover, mice over-expressing
DNase I showed a reduction in auto-antibodies but were not
protected from developing lupus nephritis (37).
We believe that the lack of effect of DNase I treatment in SLE
may be related to its internalization into cells and to the increased expression of cell surface Fas eventually leading to increased Fas-mediated apoptosis. Therefore, we think that
DNase I is not a beneficial therapy in SLE because of its ability
to increase cell susceptibility to apoptosis. On the contrary, this
treatment could be detrimental, since CI-MPR is an endocytosis receptor ubiquitously expressed in any cell types and particularly abundant in phagocytes. Therefore, DNase I delivery
within cells like neutrophils and CD34+ cells may even result in
exacerbation of some disease features like neutropenia and
leukopenia (1921). This aspect would be useful in cancer
therapy since DNase I increases tumor cells susceptibility to
apoptosis induced by chemotherapeutical agents (11). Such
hypothesis is sustained by the association of a SNP with low
DNase I activity with particular types of carcinoma (30, 31).
In conclusion, our findings show that DNase I enzyme
level is low in patients with SLE and that it can be considered a marker of the disease but not an indicator of disease
severity. Moreover, our data using PBMC from patients and

242 DNase I and SLE

one possible explanation for the lack of efficacy of hrDNase
I in the treatment of SLE.
Abbreviations
ANA
CI
CI-MPR
CRP
ESR
hrDNase
OR
PB
PBMC
sFAS
sFASL
SLE
SNP
TNF

anti-nuclear antibody
confidence interval
cation-independent mannose 6-phosphate/insulin-like
growth factor
C reactive protein
erythrocyte sedimentation rate
human recombinant DNase
odds ratio
peripheral blood
peripheral blood mononuclear cells
soluble Fas
soluble Fas ligand
systemic lupus erythematosus
single-nucleotide polymorphism
tumor necrosis factor

Fig. 3. Up-regulation of surface Fas expression and increased

susceptibility of CD3+ lymphocytes to apoptosis following exogeneous administration of hrDNAse I. Panel (A): Mean percentage 6 SD
of Fas-expressing CD3+ cells before (white bars) and after incubation
with 80 ng of hrDNase I (gray bars). Bars 14: cells derived from four
SLE patients. Bars 5 and 6: cells obtained from healthy donors. Mean
values of four independent experiments performed on each individual
subject are shown. PBMC from patients and controls were stained
with anti-CD3FITC and anti-CD95/FasPE mAbs. Panels (B and C):
extent of internucleosomal DNA fragmentation before (white bars)
and after (gray bars) incubation with hrDNase I. Bars 14: cells
obtained from SLE patients; bars 5 and 6: cells obtained from healthy
donors. Data represent the means 6 SDs of triplicate samples of
three independent experiments (x axis). Panel (B): apoptosis induced
by an anti-Fas mAb. Panel (C): apoptosis induced by human sFasL.

controls confirm previous data obtained with cell lines on the

ability of DNase I to modulate Fas expression and cell susceptibility to apoptosis. This finding, together with the measurements of DNase I and Fas expression ex vivo, suggests
that a similar mechanism may occur also in vivo, providing

1 Carroll, M. C. 1999. The lupus paradox. Nat. Genet. 19:3.

2 Chitrabamrung, S., Rubin, R. L. and Tan, E. M. 1981. Serum
deoxyribonuclease I and clinical activity in systemic lupus
erythematosus. Rheumatol. Int. 1:55.
3 Tew, M. B., Johnson, R. W., Reveille, J. D. and Tan, F. K. 2001. A
molecular analysis of the low serum deoxyribonuclease activity in
lupus patients. Arthritis Rheum. 44:2446.
4 Sallai, K., Nagy, E., Derfalvy, B., Muzes, G. and Gergely, P. 2005.
Antinucleosome antibodies and decrease deoxyribonuclease
activity in sera of patients with systemic lupus erythematosus.
Clin. Diagn. Lab. Immunol. 12:56.
5 Macanovic, M. and Lachmann, P. J. 1997. Measurement of
deoxyribonuclease I (DNase) in the serum and urine of systemic
lupus erythematosus (SLE)-prone NZB/NZW mice by a new radial
enzyme diffusion assay. Clin. Exp. Immunol. 108:220.
6 Napirei, M., Karsunky, H., Zevnik, B., Stephan, H., Mannherz, H.
G. and Moroy, T. 2000. Features of systemic lupus erythematosus
in DNase I deficient mice. Nat. Genet. 25:177.
7 Yasutomo, K., Horiuchi, T., Kagami, S. et al. 2001. Mutation of
DNase I in people with systemic lupus erythematosus. Nat. Genet.
28:313.
8 Polzar, B., Peitsch, M. C., Loos, R., Tschopp, J. and Mannherz, H.
G. 1993. Overexpression of DNase I transfected into COS-cells: its
distribution during apoptotic cell death. Eur. J. Cell Biol. 62:397.
9 Polzar, B., Zanotti, S., Stephan, H. et al. 1994. Distribution of
deoxyribonuclease I in rat tissue and its correlation to cellular
turnover and apoptosis. Eur. J. Cell Biol. 64:200.
10 Peitsch, M. C., Polzar, B., Stephan, H. et al. 1993. Characterization
of the endogenous deoxyribonuclease involved in nuclear DNA
degradation during apoptosis (programmed cell death). EMBO J.
12:371.
11 Oliveri, M., Daga, A., Cantoni, C., Lunardi, C., Millo, R. and
Puccetti, A. 2001. DNase I mediates internucleosomal DNA
degradation in human cells undergoing drug-induced apoptosis.
Eur. J. Immunol. 31:743.
12 Oliveri, M., Daga, A., Lunardi, C., Millo, R. and Puccetti, A. 2004.
DNase I behaves as a transcription factor which modulates Fas
expression in human cells. Eur. J. Immunol. 34:273.
13 Jodo, S., Pidiyar, V. J., Xiao, S. et al. 2005. Fas ligand (CD178)
cytoplasmic tail is a positive regulator of Fas ligand-mediated
cytotoxicity. J. Immunol. 174:4470.
14 Knipping, E., Krammer, P. H., Onel, K. B., Lehman, T. J., Mysler, E.
and Elkon, K. B. 1995. Levels of soluble Fas/APO-1/CD95 in
systemic lupus erythematosus and juvenile rheumatoid arthritis.
Arthritis Rheum. 38:1735.
15 Mc Nally, J., Yoo, D. H., Drappa, J. et al. 1997. Fas ligand
expression and function in systemic lupus erythematosus. J.
Immunol. 159:4628.

Downloaded from http://intimm.oxfordjournals.org/ by guest on November 26, 2014

References

DNase I and SLE 243

27 Shin, H. D., Park, B. L., Kim, L. H., Lee, H. S., Kim, T. Y. and Bae,
S. C. 2004. Common DNase I polymorphism associated with
autoimmunity production among systemic lupus erythematosus
patients. Hum. Mol. Genet. 13:2343.
28 Fujihara, J., Takatsuka, H., Kataoka, K., Xue, Y. and Takeshita, H.
2007. Two deoxyribonuclease I gene polymorphisms and correlation between genotype and its activity in Japanese population.
Leg. Med. 9:233.
29 Bodano, A., Gonzales, A., Ferreiros-Vidal, I. et al. 2006.
Association of a non-synonymous single-nucleotide polymorphism of DNASEI with SLE susceptibility. Rheumatology 45:819.
30 Tsutsumi, S., Takeshita, H., Yasuda, T., Kuwano, H. and Kishi, K.
2000. Association of DNase I phenotype 2 with colorectal
carcinoma in Japanese populations. Cancer Lett. 159:109.
31 Tsutsumi, S., Asao, T., Nagamachi, Y., Nakajiama, T., Yasuda, T.
and Kishi, K. 1998. Phenotype 2 of deoxyribonuclease I may be
used as a risk factor for gastric carcinoma. Cancer 82:1621.
32 Kumamoto, T., Kawai, Y., Arakawa, K. et al. 2006. Association of
Gln222Arg polymorphism in the deoxyribonuclease I (DNase I)
gene with myocardial infarction in Japanese patients. Eur. Heart J.
27:2081.
33 Puccetti, A., Madaio, M. P., Bellese, G. and Migliorini, P. 1995.
Anti-DNA antibodies bind DNase I. J. Exp. Med. 181:1797.
34 Silvestris, F., Williams, R. C., Calvari, N. et al. 2002. Serum
elevations of soluble Fas (CD95(apo-I) concur in deregulating
T cell apoptosis during active lupus disease. Clin. Exp. Med. 2:13.
35 Silvestris, F., Granello, D., Tucci, M., Cafforio, P. and Dammacco, F.
2003. Enhancement of T cell apoptosis correlates with increased serum levels of soluble Fas (CD95/Apo-I) in active lupus.
Lupus 12:8.
36 Davie, J. C., Manzi, S., Yarboro, C. et al. 1999. Recombinant
human DNase I (rhDNase I) in patients with lupus nephritis. Lupus
8:68.
37 Manderson, A. P., Carlucci, F., Lachmann, P. J. et al. 2006. The in
vivo expression of actin/salt-resistant hyperactive DNase I inhibits
the development of anti-ssDNA and anti-histone autoantibodies in
a murine model of systemic lupus erythematosus. Arthritis Res.
Ther. 8:R68.

Downloaded from http://intimm.oxfordjournals.org/ by guest on November 26, 2014

16 Courtney, P. A., Crockard, A. D., Williamson, K., McConnell, J.,

Kennedy, R. J. and Bell, A. L. 1999. Lymphocyte apoptosis in
systemic lupus erythematosus: relationships with Fas expression,
serum soluble Fas and disease activity. Lupus 8:508.
17 Nozawa, K., Kayagaki, N., Tokano, Y., Yagita, H., Okumura, K. and
Hasitomo, H. 1997. Soluble Fas and soluble Fas ligand in
rheumatic diseases. Arthritis Rheum. 40:1126.
18 Xue, C., Lan-Lan, W., Bei, C., Jie, C. and Wei-Hua, F. 2006.
Abnormal Fas/FasL and caspase-3-mediated apoptotic signaling
pathways of T lymphocyte subset in patients with systemic lupus
erythematosus. Cell. Immunol. 239:121.
19 Papadaki, H. A., Boumpas, D. T., Gibson, F. M. et al. 2001.
Increased apoptosis of bone marrow CD34(+) cells and impaired
function of bone marrow stromal cells in patients with systemic
lupus erythematosus. Br. J. Haematol. 115:167.
20 Courtney, P. A., Crockard, A. D., Williamson, K., Irvine, A. E.,
Kennedy, R. J. and Bell, A. L. 1999. Increased apoptotic
peripheral blood neutrophils in systemic lupus erythematosus:
relations with disease activity, antibodies to double stranded DNA,
and neutropenia. Ann. Rheum. Dis. 58:309.
21 Shoshan, Y., Shapira, I., Toubi, E., Frolkis, I., Yaron, M. and
Merovach, D. 2001. Accelerated Fas-mediated apoptosis of
monocytes and maturing macrophages from patients with systemic
lupus erythematosus: relevance to in vitro impairment of interaction
with iC3b-opsonized apoptotic cells. J. Immunol. 167:5963.
22 Xu, L., Zhang, L., Yi, Y., Kang, H. K. and Datta, S. K. 2004. Human
lupus T cells resist inactivation and escape death by upregulating
COX-2. Nat. Med. 10:411.
23 Tan, E. M., Cohen, A. S., Fries, A. et al. 1982. The 1982 revised
criteria for the classification of systemic lupus erythematosus.
Arthritis Rheum. 25:1271.
24 Bombardier, C., Gladman, D. D., Urowitz, M. B., Caron, D. and
Chang, C. H. 1992. Derivation of SLEDAI: a disease activity index
for lupus patients. Arthritis Rheum. 35:630.
25 Tsukumo, S. and Yasutomo, K. 2004. DNase I in the pathogenesis
of systemic lupus erythematosus. Clin. Immunol. 113:14.
26 Martinez Valle, F., Balda, E., Ordi-Ros, J. and Vilardell-Tarres, M. 2008.
DNase I and systemic lupus erythematosus. Autoimmun. Rev. 7:359.