apoptotic proteins, Fas, FasL, SLE, apoptosis, DNA damage, lymphocytes, Abdelmarouf H. Mohieldein

1. Proceedings of the 2006 WSEAS Int. Conf. on Cellular & Molecular Biology, Biophysics & Bioengineering, Athens, Greece, July 14-16, 2006 (pp58-62)
The system Fas/Fas-L in SLE
NATALIA BELUSHKINA1, ABDELMAROUF MOHIELDEIN2, ULIANA PETROVA3.
1,2
Department of Biochemistry, 3Deparment of immunology
1
Sechenov Moscow Medical Academy, Research Institute of Molecular Medicine, 2Omdurman Islamic
University, 3Speransky Pediatrics’ Hospital
1
Moscow 119881, M.Trubetskaya, 8/2. 2Khartoum 11111; P.O.Box 11414.
1,3
RUSSIA, 2SUDAN.
1
belushkina@mmascience.ru; 2mabdelmarouf@hotmail.com; 3uliana6@yahoo.com
Abstract: - We found significant increase of expression Fas-L on surface lymphocytes from patients with SLE
compared to normal controls (2.4* ± 1.3 vs 1.7 ± 1.1 respectively). This was found to be depending on disease
activity of SLE. However, there was a tendency to increase Fas expression on a surface of lymphocytes depending
on the degree activity of SLE. On all lymphocytes subsets CD3, CD4, CD8, and CD 19, percentages of Fas
expression in SLE were significantly higher when compared to normal controls.
There was a slight increase in soluble Fas in patients with SLE when compared to normal healthy
donors (1.6±0.2ng/ml versus 1.3±0.1ng/ml respectively). Moreover, we found an inverse correlation between sFas
and disease activity in such way that the first stage of disease characterized by the highest level of sFas compared
to second or third stages of the disease (1.8±0.1; 1.4±0.1; 1.3±0.02 respectively). Furthermore, our results showed a
positive correlation (r =0.5) between plasma sFas and percentage of lymphocytes Fas expression in patients with
SLE.
Key-Words: - Fas, Fas ligand, soluble Fas, SLE, lymphocytes subsets.
Fas-L is a type II membrane protein i.e. with an
1. Introduction amino-terminal cytoplasmic and a carboxyl-terminal
extracellular region [1]. It is a member of the TNF
The pathogenesis of systemic lupus
erythematosus (SLE) is unclear; however, recently family which includes TNF-α, α- and β-chains of
much attention has been devoted to the role of lymphotoxin (LT), CD40 ligand, and CD30 ligand
apoptosis in the pathogenesis of SLE. A putative role [6]. Fas-L is predominantly expressed on the surface
of apoptosis in the pathogenesis of human SLE was of activated T lymphocytes, natural killer (NK) cells
especially supported by a report by Emlen et al [3] [1].
describing accelerated in vitro apoptosis of SLE Fas, is type I transmembrane glycoprotein, a
lymphocytes in contrast to lymphocytes from normal member of the tumor necrosis factor (TNF) family
donors. that include tumor necrosis factor (TNF) receptor,
In an organism there are protective nerve growth factor receptor, CD40, CD27, CD30,
mechanisms that preventing synthesis of and others [1, 11]. Fas can occur as both a cell-
autoantibodies. These are processes of negative and surface and a soluble protein, with the soluble form
positive selection of lymphocytes in central and of Fas (sFas) protein being generated by alternative
peripheral organs of the immune system. The mRNA splicing [9].
deletion of these autoreactive lymphocytes through The sFas represents truncated forms of the
negative selection occurs via apoptosis. membrane-bound receptors that can bind ligand in a
One of the major routes which trigger way similar to that of their membrane- bound
apoptosis is the Fas pathway. The Cross-linking of counterparts. As a result, these receptors play an
Fas by Fas ligand (Fas-L) triggers apoptotic cell important role in regulation of normal receptor
death with the characteristic cytoplasmic and nuclear activity and autoimmune diseases.
condensation and DNA fragmentation [4].

2. Proceedings of the 2006 WSEAS Int. Conf. on Cellular & Molecular Biology, Biophysics & Bioengineering, Athens, Greece, July 14-16, 2006 (pp58-62)
Defect in the mechanism of negative selection 2.4 Determination soluble Fas (sFas) in
of lymphocytes, in particular - occurred in patients plasma by ELISA method
with autoimmune diseases who have soluble form The amounts of sFas in plasma were
Fas (sFas) in blood [2], apparently, may play a role in determined by a sandwich enzyme linked
change of regulation apoptosis through system immunosorbent assay (ELISA) using monoclonal
Fas/Fas-L [5]. antibodies CLB-CD95/2 and CLB-CD95/6.
Microtitre plates were coated with 100 µl/well
antibodies CLB-CD95/2. The process was blocked
by addition 100 µl PBS with addition of 2 %
2. Material and methods skimmed milk solution, incubated for 30 minutes at
2.1 Isolation of mononuclear leukocytes from room temperature. Samples dissolved 10 times in the
human peripheral blood ELISA-buffer (HPE - high performance ELISA).
Sterile, disposable 15 ml plastic tubes were Standard samples sFas dissolved in HPE
used for centrifugation. To each test–tube was added (concentration from 1,000 ng/ml up to 2ng/ml). 100
Ficoll gradient solution followed by 6 ml heparinised µl of each samples and a standard solution added
human blood obtained by venepuncture. 10µl biotinylated antibodies CLB-CD95/6; then add
The tubes were centrifuged at 400 x g for 30 to titre plate wells and incubated 2 hours at room
minutes at room temperature. Mononuclear temperature. Followed by reaction with horseradish
leukocytes concentrated as a ring on the border peroxidase-conjugated streptavidin (100 µl / cell).
between plasma band and isolation solution. The analysis of concentration sFas in probes carried
out on the color reaction developing after addition of
2.2 Analysis of lymphocyte subsets 0.1 mg / ml tetra-methylbenzidine and H2O2 at 0.11
The analysis of lymphocyte subpopulations M/L Na-acetate (pH =5.5) for 10 minutes (Which
carried out by flow cytometry - the analyzer and the catalyzes the conversion of the chromogenic
sorter of cells is EPICS XL-2 (“Beckman coulter”, substrate tetra-methylbenzidine from a colorless to a
USA). Suspended lymphocytes incubated with yellow solution after the addition of a stopping
monoclonal antibodies from Sorbent company reagent). Development of color reaction stopped by
(Russia). We used the fluorescent label addition 100 µl 2M H2SO4, measurement at wave
phycoerythrin conjugated monoclonal antibody (CD- length 450 nm on « Titertek Multiskan » (USA).
3, CD-4, CD-8, and CD-19).
2 µl of the required monoclonal antibodies
added to 1x106 mononuclear cells suspended in 50µl
of culture medium containing 0.1 % sodium azide. 3. Results
Incubated for 30 minutes in darkness at 4 0C. Then 3.1 Estimation level expression Fas-L / Fas in
twice wash with 200 µl icy solution (PBS with of 0.1 leukocytes peripheral blood in patients with
% sodium azide). The cells fixed with 0.1 ml of 1 % SLE
paraformaldehyde and then analyzed. Expression Fas-L on the surface of
lymphocytes in patients with SLE was significantly
2.3 Determination Fas/Fas-L in leukocytes increased in comparison with healthy donors (2.4* ±
Primary antibodies to human Fas (clone DX2, 1.3 vs 1.7 ± 1.1 respectively). (Table 1). Typical
mouse IgG1, PharMigen, USA), and to human Fas-L histograms of distribution Fas and Fas-L on a surface
(clone NOK-1, mouse IgG1, PharMigen, USA) were of cells are presented in Fig. 1.
used. Secondary antibodies of a goat to mouse IgG1, It is known that, the level expression Fas-L on
conjugated FITC, used in dilution 1:100 with (PBS , Т-lymphocytes is increased during their activation
0.5 % gelatin, and 0.1 % sodium azide). The cells and is stimulated by interleukin-2 and interferon- γ
fixed with 1 % paraformaldehyde, analyzed by flow [4]. The high level of expression Fas-L on
cytometry Epics XL (Beckman Coulter). lymphocytes can indicate activation of lymphocytes.
Increased expression Fas-L can commit Fas-
expressing cells to apoptosis.

3. Proceedings of the 2006 WSEAS Int. Conf. on Cellular & Molecular Biology, Biophysics & Bioengineering, Athens, Greece, July 14-16, 2006 (pp58-62)
We observed no significant difference in Table 2: Dependence of expression Fas/Fas-L on
percentage expression Fas on lymphocytes in patients surface lymphocytes.
with SLE when compared to healthy donors (30.8 ± Stage activity % lymphocytes (X + SD)
5.6% vs 26.1 ± 4.1). (Table 1). of SLE Fas Fas- L
I (n=18) 26.3 ± 4.9 2.1 ± 1.6
Table 1. Expression Fas / Fas-L on the surface of II (n=6) 27.7± 4.7 2.0 ± 1.8
freshly lymphocytes and granulocytes peripheral III (n=14) 32.8* ± 5.6 4.3* ± 3.3
blood. * P<0.05 in comparison with values first stage SLE
Group (%)Lymphocytes (X + SD) disease activity.
investigated Fas Fas-L
Healthy donors 26.1 ± 4.1 1.7 ± 1.1
(n=11)
3.2 Expression Fas in various lymphocyte
Patients with 30.8 ± 5.6 *
2.4 ± 1.3 subpopulations
SLE (n=38) The percentages of Fas expression on all
* P<0.05 in comparison with values for healthy lymphocytes subsets CD3, CD4, CD8, and CD 19
donors. lymphocytes in SLE were significantly higher when
compared to normal controls (Table3).
Table3. Expression CD95 (Fas) in various
lymphocyte subpopulations.
lymphocyte subpopulations, %
Group
investigated CD3 CD4 CD8 CD19
Healthy
donor 7.7±1.5 6.3±1.2 5.9 ±1.3 6.9±2.7
(n=12)
Patients
with SLE, 17.3*±3.9 20.4*±7.0 16.2*±3.02 21.0*±5.1
(n=14)
* P<0.05 in comparison with values for healthy
donors.
Fig. 1: Analysis expression Fas-L (3) and Fas (4) by
flow cytometry method. 3.3 Determination soluble sFas in plasma of
1) Lymphocytes isolated from blood of patients with patients with SLE
SLE, 2) Granulocytes isolated from blood of patients We found out that, there was a slight increase
with SLE, 3) Expression of Fas-L on surface of in soluble Fas in patients with SLE (n=46) when
lymphocytes, 4) Expression of Fas on surface of compared to normal healthy donors (n=23)
lymphocytes. X- axis: intensity fluorescence, Y- axis: (1.6±0.2ng/ml versus 1.3±0.1ng/ml. respectively).
number of cells. Shaded area display Fas-L or Fas, (Fig.2).
while non-shaded area display control.
Moreover, the high level of expression Fas-L
on lymphocytes was found to be depending on
activity of SLE disease (Table 2). The third stage
activity of SLE characterized by the highest
percentages of expression Fas and Fas-L on
lymphocytes which can indicate activation of
lymphocytes.
Fig.2. Value of concentrations soluble sFas in
patients with SLE (2) in comparison with healthy
donors (1).

4. Proceedings of the 2006 WSEAS Int. Conf. on Cellular & Molecular Biology, Biophysics & Bioengineering, Athens, Greece, July 14-16, 2006 (pp58-62)
3.4 Correlation between soluble sFas in lymphocytes and its dependence on disease activity
plasma of patients with SLE and SLE disease of SLE indicate activation of lymphocytes, since
activity peripheral resting T cells virtually do not express Fas
We investigated correlation between level ligand and express moderate amount of Fas; while
soluble Fas (sFas) and SLE disease activity. There activated peripheral T cells express abundant
was inverse correlation between sFas and disease quantities of both Fas and Fas ligand [7]. Increased
activity in such way that the first stage of disease expression Fas-L can commit Fas-expressing cells to
characterized by the highest level of sFas compared apoptosis which indicate excessive apoptosis in
to second or third stages of the disease (1.8±0.1; lymphocytes in SLE patients that promoting
1.4±0.1; 1.3±0.02 respectively). occurrence of autoantigens in plasma of patients with
SLE.
3.5 Correlation between expression Fas and Thus, it was observed the participation of Fas
receptor in pathogenesis SLE through activation of
soluble sFas in plasma of patients with SLE
apoptosis cells. The increased apoptosis lymphocytes
Moreover, we found a positive correlation (r
agreed with lymphopenia clinically diagnosed in
=0.5) between plasma sFas and percentage of
patients with SLE.
lymphocytes Fas expression in patients with SLE
Furthermore, since signaling through Fas can
(Fig.3).
induced apoptosis, this indicates that Fas seems to be
It is possible to assume that, expression Fas
functional in SLE patients, in contrast to MRL/lpr
increased at the moment of activation lymphocytes.
mice i.e., it seems that signaling through Fas is
Thus the increased level sFas in patients SLE
normally controlled and regulated in SLE cells [8].
explained the proteolytic cleavage of transmembrane
The inverse correlation between sFas and
forms Fas or alternative splicing. This can be the
disease activity can be explained by the opportunity
mechanism of regulating function of Fas receptor.
of sFas to bind membrane-bound Fas-L, which its
expression increased depending upon disease activity
at the same time the ELISA method registered the
presence of free sFas in plasma after sedimentation
of cellular elements of blood. Moreover, renal
disease, the characteristic manifestation for the third
stage of SLE, can influence circulation of proteins of
the small size and their level in plasma. Proteinuria,
in turn can result in exit of sFas from circulation [10].
Fig.3. Dependence correlation of concentration sFas References:
in plasma patients with SLE upon content activated [1] Albanese J., Sarkis M., Maria K., et al.,
lymphocytes. X axis- concentration sFas (ng/ml), Y Biologically Active Fas Antigen and Its Cognate
axis-percentage content activated lymphocytes Ligand Are Expressed on Plasma Membrane-Derived
determined by content Fas (X±SD). Extracellular Vesicles, Blood, Vol. 91. No. 10, 1998,
pp. 3862-3874.
[2] Cheng J., Zhou T, Protection from Fas-mediated
4. Conclusion apoptosis by a soluble form of Fas molecule, science.
Vol. 263. 1994, pp. 1759-1762.
It is known that, the level expression Fas-L on
Т-lymphocytes is increased during their activation [3] Elmen W., Neibur J., Kadera R, Accelerated in
and is stimulated by interleukin-2 and interferon- γ. vitro apoptosis of lymphocytes from patients with
In B-lymphocytes expression Fas-L is increased as a systemic lupus erythematosus , J. Immunol, Vol.
result of interaction of B–cells with activated T- 152, 1994, pp. 3685-3692.
lymphocytes or macrophages through system [4] Itoh, N., Yonehara S., Ishii A., et al, The
CD40/CD40L. The high level of expression Fas-L on polypeptide encoded by the cDNA for human cell

5. Proceedings of the 2006 WSEAS Int. Conf. on Cellular & Molecular Biology, Biophysics & Bioengineering, Athens, Greece, July 14-16, 2006 (pp58-62)
surface antigen Fas can mediate apoptosis, Cell, [9] Nambu Y., Steven J H., Rehemtulla A.,
Vol.6, 1991, pp. 233-243. Daniel et al., Lack of Cell Surface Fas/APO-1
[5] Krammer P. H, CD95`S deadly mission in the Expression in Pulmonary Adenocarcinomas, J.
immune system, Nature, Vol. 407, 2000, pp. 789- Clin. Invest. Vol. 101, No. 5, 1998, pp. 1102-
795. 1110.
[6] Liles WC., Kiener PA., Ledbetter JA., et al., [10] Van Lopik T., Bijl M., Hart M., et al., Patients
Differential expression of Fas (CD95) and Fas ligand with systemic lupus erythematosus with high plasma
on normal human phagocytes: implications for the levels of sFas risk relapse, J Rheumatol,. Vol.26,
regulation of apoptosis in neutrophils, J. 1999, pp. 60-67.
Experimental Medicine, Vol. 184, 1996, pp. 429-
440.
[11] Zhang J., Beiyang MM., Alan F.,
Characterization of a Novel Cis-element That
[7] Mountz J.D., Wu J., Cheng J., Zhou T, Regulates Fas Ligand Expression in Corneal
Autoimmune disease: A problem of defective Endothelial Cells, J Biol Chem., Vol. 274, Issue
apoptosis, Arthritis Rheum., Vol. 37, 1994, pp. 1415- 37, 1999, pp. 26537-26542.
1420.
[8] Nagata S., Suda T, Fas and Fas ligand: lpr and
gld mutations, Immunol Today, Vol. 16, 1995, pp. 39.