ARTICLE
Auteur(s) : Samar Abdallah M Salem1, Hanan Mohamed
Farouk2, Afaf A Mostafa3, Iman M Aly
Hassan3, Wesam M Osman4, Hebat-Allah Ahmed
Al-Shamy5, Naglaa Youssef M Assaf5
1Department of Dermatology
and Venereology
2Department of Internal Medicine
3Department of Clinical Pathology
4Department of Pathology
5Department of Rheumatology
and Rehabilitatoin, Faculty of Medicine, Ain Shams
University, Cairo, Egypt
accepté le 17 Août 2009
Systemic lupus erythematosus (SLE) is a systemic autoimmune
disease characterized by loss of self tolerance and chronic
inflammation in organs including the skin, kidney, brain and joints
[1], with the presence of autoantibodies directed against nuclear
and cytoplasmic antigens [2]. Photosensitivity is one of the
characteristics of the disease and most cutaneous lesions occur in
light exposed areas. It can be triggered by exposure to sunlight,
which can even induce SLE activity and systemic manifestations
[3].
Apoptotic cells are suggested as one of the major factors
inducing cutaneous and inflammatory lesions and autoimmunity [4].
They are increased in LE skin lesions [5]. Increased accumulation
of apoptotic cells, due to an increased rate of apoptosis,
decreased elimination of apoptotic cells, or both [6], can
challenge the immune system with immunogenic self antigens that
have been modified during apoptosis and are thus important factors
in the development of inflammatory lesions [7]. Upon phagocytosis
by macrophages, secondarily necrotic apoptotic cells will initiate
proinflammatory responses, further amplifying the inflammatory
process [8].
Increased lymphocyte apoptosis, mainly in healthy T cell
subsets, has a pathogenic role in SLE with an increased percentage
in peripheral blood of active compared to inactive patients and
resulting in accumulation of autoreactive T lymphocytes [9,
10].
One of the first events in apoptosis is the exposure of
phosphatidyl serine (PS) at the outer surface of the cell membrane.
Binding of labled annexin V has become one of the standard methods
to detect apoptotic cells in peripheral blood [11].
Neopterin is primarily secreted by stimulated macrophages
modulating its cytotoxicity [10, 12]. It induces apoptosis mediated
by reactive oxygen species (ROS) intermediates in various cell
lines [13], and its serum levels correlate positively with an
increased percentage of apoptotic lymphocytes (AL) [9]. Complement
factors are important in the clearance of late apoptotic cells
[14].
Keratinocyte exposure to ultraviolet light induces DNA damage
followed by protein 53 (p53) expression, an apoptotic protein which
plays a role in the induction of UV irradiatd apoptosis of
keratinocytes [15].
As skin lesions in SLE are characterized by apoptosis, which may
induce systemic disease activity, the aim of this work was to
differentially investigate the relationships of keratinocyte and
lymphocyte apoptosis and macrophage function to disease activity
and severity in SLE patients with and without cutaneous
manifestations.
Subjects and methods
The present case control study included 50 patients who fulfilled
the American College of Rheumatology revised criteria for SLE [16].
Their ages ranged from 20-45 years. They were recruited from the
Internal Medicine Department, Dermatology, and Rheumatology
Outpatient Clinics, Ain Shams University hospital. They were
basically treated with oral prednisone and antimalarials. Nephritis
patients were additionally under immunosuppressive therapy.
Patients with evidence of other autoimmune diseases, malignancies
and infections were excluded from the study. Twenty age and sex
matched healthy volunteers were included as controls. An informed
consent was obtained from each subject. The study was approved by
Ain Shams Medical ethics committee.
The subjects were divided into 3 groups:
- Group I: Included 25 SLE patients with specific
cutaneous manifestations of the disease (malar rash on the cheeks
and discoid lesions in sun exposed areas);
- Group II: Included 25 SLE patients with no cutaneous
manifestations;
- Group III: 20 healthy volunteers as controls.
All groups were subjected to full history taking and thorough
clinical examination. Chest X-ray, echocardiography (ECG),
electroencephalography (EEG), pulmonary function tests, and CT
scans were carried out for the patients when indicated. SLE disease
activity was assessed by the systemic lupus disease activity index
(SLEDAI) score. This is a “weighted” index of 9 organ systems for
disease activity in SLE, as follows: 8 for central nervous system
and vascular, 4 for renal and musculoskeletal, 2 for serosal,
dermal, immunologic, and 1 for constitutional and hematologic. The
maximum theoretical score is 105, but in practice, few patients
have scores greater than 45 [17].
Laboratory studies
Six milliliters of blood were collected from each subject,
3 mL on EDTA for CBC (by Coulter MAXM), ESR by Westergren
method and detection of apoptotic lymphocytes by Annexin V
apoptosis detection kit (TACs Annexin V-FITC, R&D systems, Inc.
614 Mc Kinley Place N.E. Minneapolis, MN 55413, USA). Apoptotic
cells were stained according to the manufacturer’s instructions and
analyzed using FAC scan flow cytometer (coulter EPICS XL-MCL).
From the remaining 3 mL blood, serum was collected and part
of it was used for C3 and C4 assay by RID
using Diffu-plate (Bio CientificaS-A-Buenes Aires, Argentina). The
remaining serum was stored at – 70 °C for a
subsequent neopterin assay by enzyme-immunoassay kit for the
in-vitro diagnostic quantitative determination of neopterin in
human serum, plasma and urine [Neopterin ELISA (RE 59321) IBL
Immuno-biological laboratories www.IBL-Hamburg.com]. The normal
value of neopterin in serum is expected to be <
10 nmol/L.
Twenty-four hour urine was collected and used for protein assay
by Microprotein ELI. Tech using synchron Cx9 PRO clinical
system.
Skin biopsies
Four millimeter skin biopsies were taken from specific skin lesions
of SLE patients with cutaneous manifestations and from clinically
normal sun exposed skin of SLE patients without cutaneous
manifestations. Twenty normal skin biopsies were also taken from
disease-free edges of sun exposed, surgically excised, benign
epidermal nevi or from surgical cosmetic intervention of normal
skin. Formalin-fixed, paraffin embedded skin sections of all
specimens were used for H&E and p53 immunohistochemical
staining.
Immunohistochemistry
Paraffin-embedded sections (4 μm thick) from all specimens
were mounted on poly-L-lysine-coated microscopy slides for
immuno-histochemistry using monoclonal mouse p53-antibody
(sub-class IgGi -kappa, Zymed Laboratories, San Francisco, CA, US
A). Secondary antibody (4.5 μL biotinylated anti-mouse
antibody in 1 mL of 1% BSA, Dako, Carpenteria, USA) was
pipetted onto the sections. The reaction products were visualized
by the avidin biotinylated peroxide complex methods with
diaminobenzidine as the chromogen (Dako, Carpenteria, USA). The
nuclei were stained with Mayer’s hematoxylin. The slides were then
transferred through ascending ethanol series, and xylene before
mounting.
Scoring procedure for apoptotic cells
Using Olympus Soft Pro software (Tokyo, Japan), the numbers of
immunostained cells in normal and diseased skin and the absolute
number of labeled nuclei per field at ×200 magnification were
counted. This covers an area of epidermis that includes 38 ± 1.6
basal cells. At least five randomly selected fields of each of
three sections per biopsy were counted and the mean was used for
statistical analysis [18].
Scoring procedure for dermal inflammatory infiltrate
H&E sections were semi-quantitatively scored for the presence
of inflammatory cells using a score from 0 to 5. In short, vessels
in the papillary dermis were scored blindly for the presence of
perivascular inflammatory cells, in three consecutive sections: no
infiltrating cells (0), not more than two inflammatory cells (1),
not more than one perivascular layer of inflammatory cells (2), two
or three layers of inflammatory cells (3), more than three layers
of inflammatory cells (4), and more than three layers of
inflammatory cells in combination with clear progression outside
the perivascular region (5). The final score was determined by
averaging the mean vessel score of three consecutive sections.
Inflammatory lesions were defined as the presence of category 5
(see above) vessel(s) in the dermis, with inflammatory cell
infiltration of the epidermal layer coinciding with marked local
hydropic degeneration of the basal layer of the epidermis [19].
Percutaneous renal biopsies
These were carried out in indicated SLE patients with clinical and
biochemical evidence of renal involvement (19 patients in group I
and 9 in group II). Specimens were processed and stained by
H&E. Sections were examined under light microscope and
evaluated using the WHO classification of lupus nephritis and were
accordingly classified into: class I: normal, class II:
mesangioproliferative glomerulonephritis (GN), class III: focal
proliferative GN, class IV: diffuse proliferative GN, class V:
membraneous GN, and class VI: advanced sclerosing GN [20]. Activity
and chronicity indices were interpreted and scored [21].
Statistical analysis
Data collected were revised and introduced to a PC for statistical
manipulation and analysis. Mean and standard deviation were used to
describe continuous data and number and percentage for categorical
data. Two tailed unpaired t-test was used to compare two groups as
regards quantitative variables. The Spearman correlation
coefficient test was used to test the correlation between two
quantitative variables. Chi-square test (X2) was used to
compare between two categorical variables and ANOVA test for
comparison between more than two groups regarding numerical
parameters. Results were considered significant when the P value
was < 0.05. All data management and statistical manipulations
were conducted using the 12th version of statistical
package of social science (SPSS) program [22].
Results
The present study was carried out on 50 SLE patients divided into 2
groups: group I included 25 SLE patients with specific cutaneous
manifestations (22 females and 3 males with mean age ± SD of 26.0 ±
5.5 years) and group II included another 25 SLE patients without
cutaneous manifestations (22 females and 3 males with mean age ± SD
of 25.5 ± 6.1 years). The 20 healthy volunteers who served as
controls (group III) had a mean age ± SD of 26.2 ± 5.6 years. The
mean ± SD disease duration in group I was 3.9 ± 1.6 years and in
group II was 4.0 ± 1.7 years. No statistically significant
difference was detected as regards age and sex between the three
groups nor in disease duration in the first 2 groups (P > 0.05).
As regards the clinical composition and laboratory findings of
the 2 studied SLE groups; constitutional symptoms, serositis,
musculoskeletal, hematological, neurological, mucocutaneous, and
renal manifestations were detected in 80%, 24%, 72%, 72%, 60%,
100%, and 76% of patients respectively in group I while in group II
they were detected in 44%, 16%, 56%, 48%, 32%, 0%, and 36% of
patients respectively.
SLE disease activity was assessed by SLEDAI score. Its mean ± SD
was significantly higher in group I compared to group II (18.6 ±
6.0 and 8.8 ± 2.7 respectively, P < 0.001) with a higher
percentage of patients having severe disease activity in group I
(72%) than group II (8%), (P < 0.001).
The mean percentage of peripheral apoptotic lymphocytes and mean
serum levels of neopterin were significantly higher in group I
compared to groups II and III (P < 0.001) and although they were
also higher in group II than III, the difference was statistically
insignificant (P > 0.05) (table
1).
Serum C4 levels were significantly lower in group I compared to
groups II and III (P < 0.001) while, although the level was
lower in group II than III, no significant difference was found
between them (P > 0.05) (table
1).
The mean number of p53 positive keratinocytes in the skin of
group I was significantly higher than in groups II and III (P <
0.001) and so were the mean scores of p53 positive and H&E
stained dermal inflammatory infiltrates, with no significant
difference between groups II and III (P > 0.05) (table 1). In groups II and III, p53 positive cells
were seen in an extremely low number of basal keratinocytes and
were associated with mild perivascular inflammatory infiltrate
(figure 1A). The
p53 staining in group I was seen in the nuclei of a large number of
basal and suprabasal keratinocytes and to a lesser extent in the
intermediate epidermal layer (figure 1B). The p53
staining pattern was discontinuous and involved a high proportion
of basal cells along the entire section. It was also seen in the
inflammatory infiltrate in the dermis in group I (figures 2A, B). Some
macrophages were seen in the dermis engulfing apoptotic cells.
Renal biopsies showed a higher percentage of class IV and V
lupus nephritis in group I compared to group II (P < 0.001)
(figure 3) with
significantly higher mean activity and chronicity indices in the
former group (11.7 ± 3.2 and 5.7 ± 1.9 respectively) than the
latter (6.9 ± 1.1 and 2.9 ± 0.7 respectively) with (P <
0.001).
In both SLE groups studied, the mean number of p53 positive
cells in the skin showed a significant positive correlation with
the SLEDAI score (figure
4), the mean percentage of peripheral apoptotic
lymphocytes, the serum neopterin and the mean score of dermal
inflammatory infiltrate, and a significant negative correlation to
C4 levels. On the other hand, it was significantly positively
correlated with the activity and chronicity indices of renal
biopsies in group I only (P < 0.001) (table
2).
Table 1 Comparison between the three groups as regard
the main parameters studied
|
Group I☼
|
Group II*
|
Controls♦
|
F-value/ X2
|
P-value
|
|
Apoptotic lymphocytes in peripheral blood (%) Mean (SD)
|
55.3(21.4)*♦
|
25.6(8.7)☼
|
19.4(3.2)☼
|
55.20
|
< 0.001 (HS)
|
|
Serum neopterin (nmol/L) Mean (SD)
|
27.5(7.3)*♦
|
14.9(2.7)☼
|
9.4(1.1)☼
|
106.5
|
< 0.001 (HS)
|
|
C4 (mg/dL) Mean (SD)
|
13.1(3.5)*♦
|
23.4(4.8)☼
|
28.0(6.7)☼
|
72.4
|
< 0.001 (HS)
|
|
p53+ve keratinocytes (number) Mean (SD)
|
20.6(5.4)*♦
|
1.6(0.5)☼
|
1.7(0.4)☼
|
271.8
|
< 0.001 (HS)
|
|
Score of p53+ve dermal inflammatory infiltrate Mean (SD)
|
3.7(1.0)*♦
|
1.4(0.3)☼
|
1.4(0.3)☼
|
101.3
|
< 0.001 (HS)
|
|
Score of dermal inflammatory infiltrate Mean (SD)
|
5.0(1.0)*♦
|
1.9(0.4)☼
|
1.8(0.4)☼
|
120.3
|
< 0.001 (HS)
|
Table 2 Correlation between number of p53 positive
keratinocytes and the other measured parameters in the two SLE
studied groups
|
p53+ve keratinocytes
|
Group I
|
Group II
|
|
r
|
p
|
r
|
p
|
|
SLEDAI score
|
0.814
|
< 0.01 (HS)
|
0.771
|
< 0.01 (HS)
|
|
% of peripheral apoptotic lymphocyte
|
0.784
|
< 0.01 (HS)
|
0.681
|
< 0.01 (HS)
|
|
Serum neopterin (nmol/L)
|
0.724
|
< 0.01 (HS)
|
0.744
|
< 0.01 (HS)
|
|
C4 (mg/dL)
|
– 0.730
|
< 0.01 (HS)
|
– 0.892
|
< 0.01 (HS)
|
|
Dermal inflammatory infiltrate score
|
0.734
|
< 0.01 (HS)
|
0.809
|
< 0.01 (HS)
|
|
Activity index
|
0.975
|
< 0.01(HS)
|
0.389
|
> 0.05 (NS)
|
|
Chronicity index
|
0.979
|
< 0.01 (HS)
|
0.213
|
> 0.05 (NS)
|
Discussion
SLE is a systemic autoimmune disease characterized by the presence
of autoantibodies to nuclear and cytoplasmic antigens in
conjunction with a wide range of clinical manifestations [2]. The
process inducing cutaneous and systemic inflammatory lesions has
not been clearly elucidated, but it has been suggested that
apoptosis is one of the major factors involved [4]. Owing to the
triggering effect of sun light exposure on the development of skin
lesions and the induction of SLE activity and systemic disease [3],
and because of the increased accumulation of apoptotic cells in
lupus erythematosus (LE) skin [19], we aimed in this work to
investigate the relationships of keratinocyte and lymphocyte
apoptosis and macrophage function to disease activity and severity
in SLE patients with and without skin manifestation.
Despite the presence of previous studies on either keratinocyte
or peripheral lymphocyte apoptosis, their differential evaluation
and relation to disease activity and severity in SLE patients with
and without cutaneous manifestations has not been previously
reported.
The present work showed a significantly higher number of
apoptotic cells in skin of SLE patients with cutaneous
manifestations (group I) compared to those without cutaneous
manifestations (group II) and control group (group III). Apoptotic
cells were detected in basal, suprabasal and mid epidermal layers
and within the inflammatory dermal infiltrate in this group.
Increased apoptotic cells might be due to increased rate of
apoptosis, decreased elimination of apoptotic cells [23] or both
[3]. The presence of macrophages engulfing apoptotic cells in the
first group, however, denotes a non defective engulfing action of
at least some of the macrophages.
In contrast to group I, group II SLE patients showed only a few
apoptotic cells in the basal cell layer of the epidermis with no
significant difference from the control group. As biopsies in this
group were taken from sun exposed areas, our finding can be
supported by the study done by others [24] who found no increase in
UVB induced apoptosis in SLE patients showing no skin lesions.
Accumulation of apoptotic cells has been previously demonstrated
in the skin of patients with cutaneous LE [25]. Fas antigen
expression and tunnel positive nuclei (denoting apoptosis) were
found to be present in the epidermis, hair follicles and among
cells of the dermal infiltrate in different forms of LE [5].
Accumulation of apoptotic cells in SLE promotes the release of
normally sequestered antigens with resultant activation of
autoreactive T cells [23, 26]. Autoantibodies are thus produced and
bind to apoptotic cells in skin resulting in increased production
of the proinflammatory cytokine, tumor necrosis factor-α, (TNF-α)
[5] with subsequent development of inflammatory lesions [6]. T
lymphocytes in patients with SLE are more prone to apoptosis in the
presence of TNF-α than those from healthy controls [27].
In our work, the increased number of apoptotic cells was
associated with a greater dermal inflammatory infiltrate in group I
compared to groups II and III, which supports the above mentioned
explanation. In addition, UV light also contributes to inflammation
and autoimmunity by inducing chemokine production. Chemokines
mediate the recruitment and activation of autoimmune T cells,
amplifying chemokine receptor expression and leukocyte recruitment,
finally contributing to the development of a cutaneous LE phenotype
[28].
In addition to apoptotic cells in the skin, peripheral apoptotic
lymphocytes were also found to be significantly higher in group I
compared to the other two groups, whereas the difference between
groups II and III was insignificant (despite the higher percentage
of apoptotic lymphocytes in the former). This was associated with a
significantly lower level of C4 in the first group than the other
two groups.
Decreased levels of C4, as found significantly in group I,
contribute to increased levels of apoptotic cells by reducing their
uptake by monocyte derived macrophages (MDM). This is because,
normally, many receptors as well as serum components, such as
complement proteins and pentraxins, facilitate the opsonization and
silent phagocytosis of apoptotic cells and hence the maintenance of
self tolerance [14, 29, 30].
Macrophage function was determined by the serum level of
neopterin. Thus, the significantly higher serum neopterin levels in
SLE with skin manifestations found in this work denote a higher
macrophage activity in this group. This suggests an attempt of the
macrophage system to remove the excess apoptotic cells in the skin
and blood. It also points to factors other than a primary
macrophage functional defect behind the possible defective removal
of apoptotic cells (may be complement deficiency as previously
discussed). Defective removal of apoptotic bodies leads to the
release of autoantigens which can induce autoimmunity [8].
In the present study, both disease activity (measured by SLEDAI
score) and severity were higher in SLE patients with skin
manifestations than those without. In group I the main classes of
glomerulonephritis (GN) were IV and V while in group II it was
class II. This indicates more tissue injury and organ damage in the
first group. Accelerated induction of apoptosis in T cell subsets
(especially natural killer T cells which have a regulatory role on
autoreactive T cells) by signaling abnormality, causing a decrease
in cell survival molecules [31], leads to an accumulation of
autoreactive T lymphocytes, which are left unregulated. The latter
include a subset, distinct from those that augment autoantibody
production, that is necessary for the expression of severe
nephritis [32].
Although no previous studies have compared the level of
apoptotic lymphocytes in SLE patients with and without skin
manifestations, generally a positive correlation has been found in
SLE between peripheral lymphocyte apoptosis and SLE disease
activity [9].
In the present study, the significant positive correlation of
apoptotic keratinocytes in skin biopsies with the SLEDAI score,
dermal inflammatory infiltrate, apoptotic peripheral lymphocytes
and serum neopterin in both SLE groups, with its negative
correlation to serum C4 levels, justifies and supports the role of
keratinocyte apoptosis and cutaneous apoptotic lymphocytes in
triggering inflammatory responses in both types of SLE. In
addition, its positive correlation with activity and chronicity
indices of renal biopsy in SLE with cutaneous manifestations
indicates its possible contribution to internal organ damage.
It is tempting to hypothesize the following sequence of events
in triggering systemic activity in SLE with cutaneous
manifestation. UV exposure triggers apoptosis of a large number of
keratinocytes. Accumulated apoptotic cells result in inflammation
which causes cutaneous lesions in SLE patients. UV light further
perpetuates inflammation by producing proinflammatory cytokines
(e.g. TNF-α). Defective clearance of the increased apoptotic cells
additionally contributes to its accumulation. This triggers a
systemic immune response due to flooding of the immune system with
autoantigens presented on the surface of apoptotic cells, with
subsequent activation of autoreactive T cells which are left
unregulated (after apoptosis of regulatory T-cell subsets). The
distinct subset of autoreactive T cells, necessary for the
expression of severe nephritis, is also accumulated and results in
renal damage and higher disease activity. Although increased
apoptosis can occur in SLE without cutaneous lesions, the presence
of such skin lesions could possibly exaggerate and/or trigger the
systemic manifestations of the disease.
Conclusion
In conclusion, accumulation of apoptotic keratinocytes and
lymphocytes in SLE with cutaneous manifestations is associated with
a worse disease outcome. As exposure of keratinocytes to sunlight,
a potent inducer of apoptosis, is a daily event, the skin provides
an excellent model to further unravel the underlying pathogenic
mechanisms of SLE. This might provide insights needed to treat both
the cutaneous and the non cutaneous manifestations of SLE. Further
studies concerning the nature of apoptotic cells and inflammatory
cytokines released from these cells and their exact role in
triggering activity of SLE with cutaneous manifestations are
needed.
Acknowledgement
Conflict of interest: none. Financial support: none.
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