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Element in the FasL Gene Promoter Alters Fas Ligand Expression: A Candidate Background Gene in African American Systemic Lupus Erythematosus Patients1
* Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama, Birmingham, AL 35294;
Laboratory of Medical Biochemistry, Picower Institute for Medical Research, Manhasset, NY 11030;
Department of General Surgery, Rush Presbyterian St. Luke’s Medical Center, Chicago, IL 60612; and
Epidemiology Branch, National Institute on Environmental Health Sciences, Research Triangle Park, NC 27709
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(C/EBP). Electrophoretic mobility shift and supershift assays
confirmed that this element binds specifically to C/EBP and
demonstrated that the two alleles of this element have different
affinities for C/EBP. In luciferase reporter assays, the -844C
genotype had twice the basal activity of the -844T construct, and
basal expression of Fas ligand (FasL) on peripheral blood fibrocytes
was also significantly higher in -844C than in -844T homozygous
donors. FasL is located on human chromosome 1q23, a
region that shows linkage to the systemic lupus autoimmune phenotype.
Analysis of 211 African American systemic lupus erythematosus patients
revealed enrichment of the -844C homozygous genotype in these systemic
lupus erythematosus patients compared with 150 ethnically matched
normal controls (p = 0.024). The -844C homozygous
genotype may lead to the increased expression of FasL, to altered
FasL-mediated signaling in lymphocytes, and to enhanced risk for
autoimmunity. This functionally significant SNP demonstrates the
potential importance of SNPs in regulatory regions and suggests that
differences in the regulation of FasL expression may contribute to the
development of the autoimmune phenotype. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast, recent data have demonstrated that FasL can deliver costimulatory signals (9). The balance of costimulatory and death signals varies with cell type and the stage of the immune response. Furthermore, the efficient handling of the apoptotic cells is also critical in the development in autoimmunity including systemic lupus erythematosus (SLE) (10, 11, 12), and accelerated Fas-mediated apoptosis has been implicated in the death of lymphocytes and monocytes from SLE patients (13, 14, 15, 16).
Because of the role of Fas and FasL in contributing to murine
autoimmune phenotypes and because human FasL lies in a
linkage region on chromosome 1 likely to play an important role in SLE
(17, 18), we considered FasL as a potential
candidate gene contributing to human autoimmunity. FasL
consists of four exons and spans 
8 kb on chromosome 1q23
(19). The FasL coding region has been screened
by single-strand conformation polymorphism, but mutations have been
implicated in only a single SLE patient (20). However,
Murga et al. (21) have induced an autoimmune phenotype by
enhancing T cell proliferation through genetic modification of the E2F2
transcription factor. The transcriptional regulation of FasL expression
in immune system cells involves NF-
B, NF-AT, early growth response
gene-3, and several other uncharacterized elements in the
FasL promoter (22, 23, 24, 25, 26). However, little
information is available about genetic variations of the human
FasL promoter region that may affect FasL expression in both
T cells and other cells. Therefore, we analyzed the promoter of
FasL and have identified a naturally occurring, functionally
significant single-nucleotide polymorphism (SNP) in a newly recognized
CAAT/enhancer-binding protein (C/EBP) element in the enhancer
region of FasL. This C/EBP element affects FasL protein
expression in vitro and ex vivo and is associated with SLE in African
American patients. These data suggest that genetic variation in
promoter elements of key immunoregulatory molecules may contribute to
an autoimmune diathesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anticoagulated peripheral blood was obtained from healthy normal volunteers, from rheumatoid arthritis patients fulfilling the criteria of the American College of Rheumatology for rheumatoid arthritis (27), and from SLE patients fulfilling the revised American College of Rheumatology criteria for SLE (28). SLE patients and controls were recruited as part of the University of Alabama-based DISCOVERY cohort and as part of the Carolina Lupus Study, a population-based case-control study of risk factors for SLE. The human studies were reviewed and approved by the Institution Review Board, and all donors provided written informed consent.
Reagents
Anti-CD3 mAb was purified from the culture supernatant from OKT3
hybridoma (American Type Culture Collection, Manassas, VA). Mouse
anti-human FasL mAb (NOK-1), anti-CD40 mAb, and FITC-conjugated
anti-mouse IgG1 mAb were purchased from BD PharMingen (San Diego,
CA). PMA, LPS, and recombinant human IL-6 (IL-6) were obtained from
Sigma-Aldrich (St. Louis, MO). Ionomycin was purchased from Calbiochem
(La Jolla, CA). Rabbit anti-C/EBP polyclonal IgG was obtained
from Santa Cruz Biotechnology (Santa Cruz, CA). Transfection reagent
DMRIE-C was obtained from Life Technologies (Gaithersburg,
MD). Reagents for luciferase reporter assays were purchased from
Promega (Madison, WI).
Nucleic acid isolation
Human genomic DNA was isolated using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN). Briefly, 300 µl of blood were lysed in 900 µl of the red blood lysis solution, and the leukocytes were pelleted and lysed with 300 µl of cell lysis solution. The RNase A solution was added to the cell lysate and incubated at 37°C for 15 min. Proteins were precipitated by the addition of 100 µl of protein precipitation solution. DNA in the supernatant was precipitated with 300 µl of 100% isopropanol and washed once with 70% ethanol.
RT-PCR and cDNA sequencing
To facilitate heterozygote detection, a dye primer strategy was adopted for fluorescence-based automated cycle sequencing of PCR products on an ABI 377 (ABI PRISM Dye Primer Cycle Sequencing -21 M13 FS and M13REV FS Ready Reaction Kits; Applied Biosystems, Foster City, CA). For FasL RT-PCR, sense primer 5'-TGT AAA ACG ACG GCC AGT CCT GAC TCA CCA GCT GCC AT-3' with M13 forward sequence tag (underlined letters) anneals to nucleotide positions 68 to 87, and antisense primer 5'-CAG GAA ACA GCT ATG ACC GGA AAG AAT CCC AAA GTG-3' with M13 reverse sequence tag (underlined letters) anneals to positions 938 to 955 (GenBank accession number U08137). PCR was performed in a 9600 PCR System with 2 µl of cDNA synthesized with the SuperScript Preamplification System (Life Technologies), 300 nM concentrations of each primer, 200 µM concentrations of dNTPs, 1.5 mM MgCl2, and 2.5 U of Taq DNA polymerase in a 100-µl reaction volume starting with 95°C for 5 min, 35 cycles of denaturing at 94°C for 30 s, annealing at 58°C for 45 s, and extension at 72°C for 1 min, with a final extension at 72°C for 7 min. The PCR product was purified with the QIAquick Gel Extraction Kit (Qiagen, Chatsworth, CA). All PCR products were sequenced in both directions.
PCR amplification and sequencing of FasL promoter region
The FasL promoter region was amplified with the sense
primer 5'-TTA TGC CTA TAA TCC CAG CTA CTC A-3' annealing to nucleotide
positions from -1032 to -1008, and antisense primer 5'-CTG GGG ATA
TGG GTA ATT GAA G-3' annealing to positions from +12 to +33 (Fig. 1
, +1 site corresponds to the A of ATG
translation start codon). The PCR was performed with 500 ng of DNA, 300
nM concentrations of each primer, 200 µM concentrations of dNTPs, 1.5
mM MgCl2, and 2.5 U of Taq DNA
polymerase in a 100-µl reaction volume starting with 95°C for 5
min, 35 cycles of denaturing at 94°C for 30 s, annealing at
58°C for 45 s, and extension at 72°C for 1 min, with a final
extension at 72°C for 7 min. All the PCR products were purified from
a 2.5% agarose gel with the QIAquick Gel Extraction Kit. The purified
PCR products were sequenced from both directions using the BigDye
terminator sequence on an ABI 377.
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The characterization of the FasL promoter was verified by dye-primer sequencing with M13 sequence-tagged primers. The sense primer, 5'-TGT AAA ACG ACG GCC AGT CCA GCC TGG GTG ACA GAG TGA-3', anneals from position -933 to -913; and the antisense primer, 5'-CAG GAA ACA GCT ATG ACC TAT AGC CCT GTT AGT GTG AAC T-3', anneals from position -416 and -495 (underlined nucleotides are M13 forward or reverse sequences). In addition, gel-purified 1 kb of PCR product was directly cloned into pGEM-T Easy Vector (Promega). At least 10 clones from each of 6 heterozygous donors were sequenced. All cloned sequences and dye-primer sequences were in agreement with cycle sequencing of genomic template using the BigDye terminator strategy.
Nuclear extract preparations
Jurkat cells (107/ml) were stimulated with 10 µg/ml LPS for 2 h or cultured on anti-CD3 mAb-coated plates for 3.5 h before nuclear extraction. Cells were washed with PBS (pH 7.4), resuspended in buffer (10 mM HEPES, 1.5 mM MgCl2, and 10 mM KCl in the presence of protease inhibitors and 100 µM DTT), and lysed by the addition of Nonidet P-40 to a final concentration of 0.1%. Nuclei were pelleted and washed in buffer A, and nuclear protein was extracted in buffer C (20 mM HEPES, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, and 0.2 mM EDTA). After the nuclear debris was pelleted, the supernatant was removed and diluted with an equal volume of buffer D (20 mM HEPES, 20% glycerol, 50 mM KCl, and 0.2 mM EDTA). Protein concentration was determined by measuring OD280.
Electrophoretic mobility shift and supershift assays
For each binding reaction, 8 µg of nuclear extract were
incubated in 1x binding buffer (4% glycerol, 1 mM
MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM
Tris-HCl (pH 7.5), and 50 µg/ml poly(dI-dC)·poly(dI-dC)) with
32P-labeled oligonucleotides in a volume of 10
µl. Binding reactions were incubated at room temperature for 30 min
with 50,000 cpm (0.1–0.5 ng) of double-stranded oligonucleotides
end-labeled with [
-32P]ATP using T4
polynucleotide kinase. Unlabeled specific or nonspecific competitor
oligonucleotides were used where indicated at a 200-fold molar excess.
Protein/DNA complexes and unbound DNA probe were resolved on 5%
nondenaturing polyacrylamide gel and visualized by autoradiography. The
following double-stranded oligonucleotide probes were used in these
experiments: C/EBP consensus, 5'-TGC AGA TTG CGC AAT CTG CA-3'
(Santa Cruz Biotechnology); nonspecific, 5'-GTG GGT GTT TGT TTG AGA-3';
C/EBP -844C allele, 5'-AAA ACA TTG CGA AAT ACA-3';
C/EBP -844T allele, 5'-AAA ACA TTG TGA AAT ACA-3'
(polymorphic nucleotide position -844 is underlined) (Fig. 1
). The
C/EBP supershift assay was conducted with specific polyclonal Abs by
following the manufacturer’s instruction (Santa Cruz
Biotechnology).
FasL reporter constructs
The FasL luciferase reporter constructs of the -844C
and -844T alleles of C/EBP were generated by cloning a
KpnI/HindIII-flanked PCR product with 1026
nucleotides of FasL promoter region into pGL3-Basic vector
(Promega). The KpnI/HindIII-flanked PCR product
was obtained by amplifying from human genomic DNA with upper primer
5'-GGC GGA GGT ACC CTA TAA TCC CAG CTA CTC AG-3'
(underlined and bold nucleotides are KpnI cutting site)
annealing at positions -1026 to -927 and lower primer 5'-GTT CCG
AAG CTT GGC AGC TGG TGA GTC AGG C-3' (underlined
and bold nucleotides are HindIII cutting site) annealing at
positions -19 to -1. The subsequent change at nucleotide position
-844 was made using a QuikChange Site-Directed mutagenesis kit
(Stratagene, La Jolla, CA). For -844T construct, sense primer 5'-AAA
TGA AAA CAT TGT GAA ATA CAA AGC AG-3' and antisense primer 5'-CTG CTT
TGT ATT TCA CAA TGT TTT CAT TT-3' were used for mutagenesis. All the
constructs were sequenced from both directions.
Transient transfection, cell stimulation, and luciferase assays
The Jurkat human leukemic T cell line was maintained in RPMI
with 10% FCS, penicillin (1000 U/ml), streptomycin (1000 U/ml), and
glutamine (2 mM). Each transient transfection experiment was conducted
with 2 x 106 Jurkat cells and 2 µg of
construct plasmid DNA plus 0.5 µg of pCMV.SPORT--galactosidase
plasmid (Life Technologies) purified with the Wizard PureFection
Plasmid DNA Purefection System (Promega) by using 4 µl of DMRIE-C
reagent (Life Technologies). Transfected cells were cultured for
18 h without stimulation, with 2 µg/ml LPS, 20 ng/ml human IL-6,
or 50 ng/ml PMA plus 1 µM ionomycin (Calbiochem), or in anti-CD3
mAb-coated wells that had been preincubated with 1 ml of 20 µg/ml
anti-CD3 mAb at 37°C for 2 h. The harvested cells were
washed twice with PBS (pH 7.4) and were lysed in 300 µl of 1x lysis
buffer (Promega). Cell debris was removed by centrifugation, and the
supernatant was used in the luciferase reporter assay using a Monolight
2010 luminometer (Promega). Luciferase light units, standardized to
-galactosidase activity, are reported as the mean of triplicate
samples.
Fibrocyte isolation and detection of FasL expression
Human fibrocytes were purified from peripheral blood as previously described (29, 30). Briefly, heparinized blood was mixed with PBS (1:1), and 8 ml were layered over 4 ml of Ficoll-Hypaque and centrifuged at 400 x g for 30 min at room temperature. The buffy coat was isolated, washed with PBS, and spun at 500 x g and then washed with PBS. Human fibrocytes were maintained in DMEM supplemented with 20% FCS (HyClone Laboratories, Logan, UT), and 1% penicillin-streptomycin-glutamine. After 7 days in culture, fibrocytes were treated with anti-CD40 mAb (2.5 µg/ml) or untreated and incubated for an additional 2 days. The adherent fibrocytes were lifted by incubation in ice cold 0.05% EDTA (Sigma) in PBS and collected for analysis of FasL expression by flow cytometry. Cells (106 aliquots) were resuspended in PBS containing 3% BSA and 0.1% sodium azide and incubated with anti-human FasL mAb for 30 min at 4°C. After two washes, the cells were incubated with FITC-conjugated anti-mouse IgG1 for 30 min. The cells were washed and fixed (R&D (Minneapolis, MN) fixative), and fluorescence data were acquired on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and analyzed using CellQuest (BD Biosciences) software.
Data analysis
TESS (Transcription Element Search Software; http://www.cbil.upenn.edu/tess/) and MatInspector (http://www.gsf.de/biodv/matinspector.html) were used to search for candidate transcription factors that might bind the regions adjacent to and including nucleotide position -844. Fisher’s exact test was used to analyze the distribution of FasL promoter genotypes and gene frequencies in SLE patients and normal controls. Differences in FasL promoter activities of various constructs were analyzed by Student’s t test. Results from FasL expression in paired samples of genotyped normal donors were analyzed by the Wilcoxon matched pairs signed rank test. The null hypothesis was rejected at the 95% confidence level (p < 0.05).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A search of SNP databases did not reveal any polymorphisms in the
FasL coding region. Therefore, we sequenced the coding
region of 51 donors (14 SLE patients, 30 rheumatoid arthritis patients,
and 7 normal subjects) and found no polymorphic sites that predict
amino acid changes within the FasL protein. Next, we amplified the
proximal 1 kb of FasL promoter region and directly sequenced
the purified PCR products (Fig. 1). As shown in Fig. 2, we observed a SNP at nt -844 within
this fragment of the FasL promoter region. In addition to
the C at position -844 found in GenBank (Z96050), we found a T allele
in our normal populations with an allele frequency of 0.82 for African
Americans (Table I). Homozygous C/C,
homozygous T/T, and heterozygous C/T donors were identified (Fig. 2).
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-binding region and allele-specific
effects on the binding affinity for the C/EBP
Computer analysis indicated that sequences surrounding nt -844
might bind members of the CCAAT/enhancer binding protein family with a
perfect match of the sequence, ATTGCGAAAT (the underlined
nucleotide is polymorphic), for the binding site of CCAAT/enhancer
binding protein (C/EBP, or NF-IL-6). C/EBP has the consensus
recognition site as 5'-T(T/G)NNGNAA(T/G)-3', and the polymorphic
nucleotide sits in the core of the C/EBP binding motif (Fig. 1).
EMSA analysis of the surrounding 18-bp genomic sequence confirmed that
the putative C/EBP motif from position -848 to -839 is indeed a
putative C/EBP binding site. Radiolabeled oligonucleotide probe
containing the putative -848 to -839 C/EBP motif binds nuclear
extract from activated Jurkat cells (Fig. 3, lane 2) and is inhibited
both by the consensus CCAAT enhancer-binding oligonucleotide
(lane 1) and by cold oligonucleotide containing the
putative C/EBP motif (lane 3). Anti-C/EBP Ab
can supershift the labeled probe/nuclear extract complexes
(lane 4), providing further evidence that C/EBP is
indeed the transcription factor that binds the region containing
position -844. Interestingly, labeled probe with the -844T allele
(lane 6) bound but appeared to have much lower
affinity for the transcription factor C/EBP than that of the -844C
allele. Taken together, these data indicate that nt -844 and the
adjacent sequences constitute a putative C/EBP element and that the
two different alleles have significantly different affinities for the
C/EBP.
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element on the promoter
activity in Jurkat cells
FasL reporter constructs differing only by the single SNP at
position -844 demonstrated significantly different promoter activities
in Jurkat cells in both basal and stimulated conditions (Fig. 4A). Under basal conditions,
the promoter activity incorporating the -844C allele was 2.4-fold
greater than that of the -844T allele (means: 16,203 vs 6,706 relative
light units for -844C vs -844T, respectively; p <
0.01). Neither human IL-6 nor LPS alone enhanced the activity of the
FasL promoter (Fig. 4B), but after stimulation with
anti-CD3, the promoter activity of the -844C allele was 2.3
greater than that of -844T allele (93,117 vs 40,187 relative light
units, respectively; p < 0.01), a difference that was
also maintained with the stimulation by PMA plus ionomycin (66,867 vs
29,594 relative light units, respectively; p < 0.01).
Therefore, in support of the EMSA data, the luciferase reporter assay
provided strong evidence that the polymorphism within the
C/EBP-binding site of the FasL promoter enhancer region
could significantly alter the promoter activity. The luciferase data
also suggest that additional regulatory elements are involved in the
response to stimulation.
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element on the expression of
FasL by human fibrocytes
Because attempts to demonstrate FasL protein expression on
peripheral T cells were unsuccessful, we examined circulating
fibrocytes as a model system for FasL expression (31). The
FasL promoter of normal donors was sequenced and donors
homozygous for either -844C or -844T were identified. Consistent with
the results obtained from the luciferase promoter activity assay, the
basal expression of FasL on fibrocytes derived from -844C homozygous
donors were 2- to 3-fold higher than that on fibrocytes from -844T
homozygous donors in eight of eight paired comparisons (Fig. 5, p < 0.01). Expression
levels stimulated in vitro by anti-CD40 were less consistent, with
four of six paired comparisons higher and the average level in -844C
homozygous donors 15% above that for -844T donor values.
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Because there is accelerated apoptosis of lymphocytes and
monocytes from patients with systemic lupus erythematosus, we
hypothesized that the -844C allele of the FasL promoter
might be associated with autoimmune disease. Therefore, we compared
genotype frequencies in 211 African American SLE patients with those of
150 ethnically matched normal subjects (Table I). Among African
Americans, the percentage of -844C homozygotes was significantly
enriched in SLE patients (9%) compared with those of corresponding
normal controls (3%), suggesting a recessive effect (Fisher’s exact
test, p < 0.024). Although the CC homozygotes in
Caucasian SLE patients were also enriched compared with those of
Caucasian normal controls, the difference was not statistically
significant.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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to 
),
all sharing high homology in the C-terminal domain, which carries a
basic DNA-binding domain and a leucine zipper motif. C/EBP was
originally identified as a mediator of IL-6 signaling, binding to
IL-6-responsive elements in the promoters of acute phase response genes
such as TNF, IL-8, and G-CSF (32, 33). IL-1 and LPS also
induce C/EBP transcription (32, 34). C/EBP plays
crucial roles in the functional regulation and homeostatic control of
the myeloid and lymphoid compartments. C/EBP-deficient mice have
altered antibacterial and tumoricidal activities reflecting defective
macrophage activation. C/EBP-deficient mice also develop a
lymphoproliferative disorder reminiscent of Fas/FasL mutations in mice.
These mice have altered CD4+ Th responses and an
expansion of the B cell compartment accompanied by high levels of
IgG-bearing cells in the lymph nodes and spleens (35, 36).
Gene expression arrays indicate that C/EBP is highly induced in
tolerant B cells, strongly suggesting a relationship with immune
tolerance (37). Given this context, our identification of
a functionally important C/EBP enhancer element in the
FasL promoter region suggests one mechanism for the crucial
role of C/EBP in the peripheral tolerance and cytotoxicity in
C/EBP-deficient mice.
Despite our understanding of the importance of FasL in immune
homeostasis, little is known about its natural transcriptional
regulation. Jurkat T cells have been widely used in the
characterization of the FasL promoter elements
(22, 23, 24, 26). The concordant results of FasL
promoter activities of -844 SNPs both in fibrocytes and in Jurkat T
cells indicate that they are suitable model cell systems for
FasL promoter characterization. Our data indicate that the
C/EBP element in the FasL promoter plays an important
role in the regulation of expression of FasL gene both in
vitro and in vivo. The polymorphism of C/EBP element significantly
affects the biological activity of FasL promoter and altered
the basal FasL expression on fibrocytes. Human fibrocytes can serve as
APCs and induce APC-dependent T cell proliferation when cultured with
specific Ag. T cell-proliferative activity induced by fibrocytes is
significantly higher than that induced by monocytes and may be
nearly as high as that induced by purified dendritic cells
(38). The expression of FasL indicates that fibrocytes may
also be involved in immune tolerance, and the difference in FasL
expression by fibrocytes could have significant effects on the
apoptosis of Fas-expressing lymphocytes and monocytes. Together with
the luciferase reporter assays which indicate that the C/EBP
polymorphism has a persistent effect on the induction levels of the
FasL expression in T cells, the polymorphism of the C/EBP element in
the FasL promoter could have significant implication in the
pathogenesis of autoimmune diseases.
Predicting the net impact of FasL promoter SNPs on an
autoimmune disease phenotype is complicated both by the capacity of
FasL to deliver costimulatory as well as proapoptotic signals and by
the observation that the Fas/FasL-related phenotype in mice is
background dependent. Individuals carrying the high activity allele of
the FasL C/EBP element could have more apoptosis of the
activated lymphocytes, monocytes, macrophages, and other cells, which
in turn might contribute the increased levels of nucleosomes found in
the circulation of SLE patients (39). Such an ongoing
source of extracellular nuclear Ags to drive more profound immune
response and to allow the formation of extra immune complexes in the
patients and may overwhelm the clearance system of the human body.
Indeed, Kovacs et al. (13) have reported the increased
expression of functional FasL in activated T cells from SLE patients
and have proposed that the elevated FasL expression could account for
the high apoptotic rate of lymphocytes in SLE patients. Alternatively,
the autocrine interaction of Fas and FasL on activated T cells
contribute to the maintenance of peripheral T cell tolerance, and FasL
expressing APCs can delete responding T cells in the periphery, leading
to Ag-specific systemic T cell tolerance (40).
Our genotyping data suggest that the higher activity allele of the
C/EBP element is associated with SLE in African Americans. This
association must be confirmed in independent case-control and
family-based association studies. Based on the established epistatic
interaction of Fas/FasL mutations with background genes in
mouse models, it will be interesting to assess the genetic associations
in humans, conditioned on other genetic effects and stratified by
different ethnic groups. These studies and studies to consider that the
-844 genotype may also be in linkage disequilibrium with other
susceptibility gene(s) in this region are currently under way.
Like other spontaneous autoimmune disease, the lupus diathesis most
likely represents the summation of multiple genetic effects, each
contributing a small risk of disease. The C/EBP element polymorphism
may play such a role in the pathogenesis of autoimmune diseases as a
disease susceptibility gene. However, it is also important to recognize
that FasL participates in more than maintenance of tolerance and in the
genesis of autoimmune disease. The integrity of sites of immune
privilege and the efficiency of immune surveillance and evasion by
cancer cells may also be affected by variation in FasL expression.
Indeed, these areas, although unexplored, may be among the most
important physiological settings for the role(s) of this promoter
variant.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert P. Kimberly, Division of Clinical Immunology and Rheumatology, University of Alabama, 429 Tinsley Harrison Tower; 1900 University Boulevard, Birmingham, AL 35294-0006. E-mail address: rpk{at}uab.edu
3 Abbreviations used in this paper: FasL, Fas ligand; SLE, systemic lupus erythematosus; SNP, single-nucleotide polymorphism; C/EBP, CAAT/enhancer-binding protein .
Received for publication February 6, 2001. Accepted for publication November 1, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-deficient mice. [Published erratum appears in 1995 EMBO J. 14:3596.]. EMBO J. 14:1932.[Medline]
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T. Sun, X. Miao, X. Zhang, W. Tan, P. Xiong, and D. Lin Polymorphisms of Death Pathway Genes FAS and FASL in Esophageal Squamous-Cell Carcinoma J Natl Cancer Inst, July 7, 2004; 96(13): 1030 - 1036. [Abstract] [Full Text] [PDF]
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) and -844T homozygous donor (
). The -844C homozygous
donors expressed significantly more FasL on the fibrocytes than the
-844T homozygous donors (Wilcoxon matched pairs signed rank test,
p < 0.01). The results indicated are averages of
the normalized mean fluorescent intensity in eight paired comparisons.
B, Representative histogram of FasL expression on human
fibrocytes. FasL expression on fibrocytes derived from two -844C
homozygous donors (dark line and dotted line on the right
side) and one -844T homozygous donor (dark line on the left
side) is shown. Shaded area, Isotype control.