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Blood, Vol. 95 No. 10 (May 15), 2000:
pp. 3176-3182
IMMUNOBIOLOGY
Deficiency of the Fas apoptosis pathway without Fas gene
mutations is a familial trait predisposing to development of autoimmune
diseases and cancer
Ugo Ramenghi,
Sara Bonissoni,
Giuseppe Migliaretti,
Simona DeFranco,
Flavia Bottarel,
Caterina Gambaruto,
Daniela DiFranco,
Roberta Priori,
Fabrizio Conti,
Irma Dianzani,
Guido Valesini,
Franco Merletti, and
Umberto Dianzani
Department of Pediatrics and Department of Biomedical Sciences and
Human Oncology, University of Turin, Turin, Italy; Department of
Medical Science, "A. Avogadro" University of Eastern Piedmont,
Novara, Italy; Chair of Allergology and Clinical Immunology, Institute
of Clinical Medicine I, "La Sapienza" University of Rome, Rome,
Italy.
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Abstract |
Fas/Apo-1 (CD95) triggers programmed cell death (PCD) and is
involved in immune response control and cell-mediated cytotoxicity. In
the autoimmune/lymphoproliferative syndrome (ALPS), inherited loss-of-function mutations of the Fas gene cause nonmalignant lymphoproliferation and autoimmunity. We have recently identified an
ALPS-like clinical pattern (named autoimmune lymphoproliferative disease [ALD]) in patients with decreased Fas function, but no Fas gene mutation. They also displayed decreased PCD response to ceramide, triggering a death pathway partially overlapping that used
by Fas, which suggests that ALD is caused by downstream alterations of
the Fas signaling pathway. Decreased Fas function is also involved in
tumor development, because somatic mutations hitting the Fas system may
protect neoplastic cells from immune surveillance. This work assessed
the inherited component of the ALD defect by evaluating Fas- and
ceramide-induced T-cell death in both parents and 4 close relatives of
10 unrelated patients with ALD. Most of them (22 of 24) displayed
defective Fas- or ceramide-induced (or both) cell death. Moreover,
analysis of the family histories showed that frequencies of
autoimmunity and cancer were significantly increased in the paternal
and maternal line, respectively. Defective Fas- or ceramide-induced
T-cell death was also detected in 9 of 17 autoimmune patients from 7 families displaying more than a single case of autoimmunity within
first- or second-degree relatives (multiple autoimmune syndrome [MAS] patients). Autoimmune diseases displayed by ALD and MAS families included several organ-specific and systemic forms. These data suggest
that ALD is due to accumulation of several defects in the same subject
and that these defects predispose to development of cancer or
autoimmune diseases other than ALPS/ALD.
(Blood. 2000;95:3176-3182)
© 2000 by The American Society of Hematology.
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Introduction |
Fas/Apo-1 (CD95), a transmembrane molecule belonging to
the tumor necrosis factor receptor (TNFR) superfamily, binds FasL (CD95L) belonging to the tumor necrosis factor (TNF)
superfamily.1,2 Fas/FasL interaction triggers programmed
cell death (PCD) in several cell types and may play a role in immune
response control, lymphocyte life span regulation, and peripheral
tolerance induction. Moreover, Fas is involved in cytotoxic T
lymphocyte (CTL) and TH1 cell cytotoxicity, which is partially due to
interaction of FasL expressed by activated cytotoxic cells with Fas
expressed by target cells. In lymphocytes, Fas triggering does not
induce PCD in resting and recently activated T cells, but the
PCD-inducing pathway is connected to Fas several days after cell activation.
In lpr/lpr mice and in patients with the autoimmune
lymphoproliferative syndrome (ALPS), inherited loss-of-function
mutations of the Fas gene have been associated with a clinical
picture characterized by nonmalignant lymphoproliferation with
lymphadenopathy or splenomegaly and peripheral expansion of T-cell
receptor (TCR  +) T cells that are double negative for
CD4 and CD8 (DN T cells), and autoimmune phenomena.1,3-9
Lpr/lpr mice develop hypergammaglobulinemia, autoantibody
production, glomerulonephritis, arthritis, and vasculitis, whereas
patients with ALPS display hemolytic anemia, thrombocytopenia, neutropenia, or their combinations, recurrent urticaria consistent with
immune vasculitis, and glomerulonephritis. Despite their shared
clinical and molecular pattern, the human and mouse diseases display
different genetic inheritance. In the mouse, expression of the disease
requires homozygous mutations of the Fas gene, whereas most
ALPS patients are heterozygous for Fas mutations. Intriguingly, their
parents who are heterozygous for the Fas mutation are generally
healthy, which suggests that either patients carry mutations in other
complementary genes or specific environmental factors are required to
trigger the disease.
In a previous work, we identified 6 unrelated patients with an
ALPS-like clinical pattern, though with no DN cell
expansion.10 T cells displayed reduced Fas capacity to
induce PCD, but no Fas gene mutation, together with decreased
PCD response to ceramide, which triggers a death pathway partially
overlapping that used by Fas. These data showed that clinical pictures
with lymphoproliferation and autoimmunity may involve not only Fas
itself, as in patients with ALPS, but also its signaling pathway, and
suggest that Fas gene mutations and DN cell expansion are not
the only signs of a defective Fas system. Because the disease displayed
by our patients did not fit the crucial parameters required to diagnose
ALPS, we operatively named it autoimmune lymphoproliferative disease (ALD).
Decreased function of Fas has also been suggested to play a role in
tumor development.11-16 Fresh tumor cells and tumor cell lines, in fact, are often resistant to Fas-induced cell death, because
they down-regulate Fas expression, secrete soluble forms of Fas, or
express a Fas molecule with decreased signaling activity. These
alterations may be ascribed to somatic mutations hitting the Fas system
in the multistep cancerogenesis process and may protect neoplastic
cells from immune surveillance. Moreover in a recent work, somatic
mutations of the Fas gene have been associated with development
of a clinical picture with lymphoma and autoimmunities.17 Development of lymphomas has also been anecdotally reported in some
families of ALPS patients.1,3-5
The aim of this work was to test the hypothesis that ALD is a genetic
disease and to evaluate whether this genetic pattern predisposes to
development of neoplasia and autoimmune diseases that are different
from typical ALPS/ALD. We found that T cells from the parents of all
the ALD patients displayed defective Fas- or ceramide-induced death (or
both). Frequencies of autoimmune diseases and death from cancer were
increased in these families in the paternal and maternal line,
respectively. Moreover, defective Fas- or ceramide-induced T-cell death
was detected in autoimmune patients from families displaying a high
frequency of autoimmunity.
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Materials and methods |
Patients
The ALD patients were those with chronic hematologic autoimmunities
(anemia, thrombocytopenia, or neutropenia), lymphadenopathy or
splenomegaly, and T-cell resistance to cell death induced in vitro by
anti-Fas monoclonal antibodies (mAb) (Figure
1) in the absence of mutations of the
Fas gene. ALD patients 1, 2, 3, 5, 6, and 7 were previously
described.10 Control families were those of 21 consecutive
children observed in the general practitioner outpatient clinic. The
main clinical data for each patient are listed below.
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| Fig 1.
Fas-induced T-cell death in ALD patients and 15 normal
donors.
Left panel shows T cells cultured for 6 or 21 days with IL-2 and
treated with anti-Fas mAb; relative cell survival was assessed after 18 hours. Right panel shows day-21 T-cells that were treated with anti-Fas
mAb. Cell apoptosis was assessed after 6 hours by annexin V staining.
Results are expressed as percentage of annexin V+ cells in
the Fas-treated well corrected for the percentage of annexin
V+ cells in the Fas-untreated well. The percentage of
annexin V+ cells in the Fas-untreated well was always less
than 10%. Results from normal donors (controls) are shown as mean ± SD.
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ALD.1 family.
Man, aged 24 years, was observed at the age of 13 for thrombocytopenia,
splenomegaly, and lymphadenopathy. Subsequently he also developed
neutropenia. Mild thrombocytopenia and neutropenia persisted after splenectomy.
ALD.2 family.
Boy, aged 5 years, was observed at 4 months of age for anemia,
thrombocytopenia, and splenomegaly. He is in remission after intravenous immunoglobulin and steroids; splenomegaly persists.
ALD.3 family.
Boy, aged 10 years, presented at age 3 years with thrombocytopenia and
neutropenia with generalized lymph node enlargement. At present, he is
in hematologic remission, but shows mild splenomegaly and adenomegaly.
ALD.4 family:
Boy, aged 12 years, was treated at the age of 7 years for
subdiaphragmatic stage IIb Hodgkin disease. Since the age of 9 he has
been followed for chronic thrombocytopenia responsive to high steroid doses.
ALD.5 family.
Girl, aged 13 years, was hospitalized at the age of 8 months for
persistent fever and substantial splenomegaly. She has always presented
anemia and high levels of IgG, which persist after splenectomy performed at the age of 12.
ALD.6 family.
Man, aged 18 years, has thrombocytopenia and large axillary and
inguinal lymph nodes. Adenopathy and thrombocytopenia persist after splenectomy.
ALD.7 family.
Boy, aged 6 years, has very large spleen and thrombocytopenia.
ALD.8 family.
Girl, aged 13 years, was admitted to our department at the age of 7 for
severe hemolytic anemia and splenomegaly. She presents recurrent
episodes of thrombocytopenia or hemolytic anemia, both responsive to a
high dose of steroids.
ALD.9 family.
Girl, aged 12 years, was observed at the age of 10 for severe
thrombocytopenia and splenomegaly. Thrombocytopenia persists after splenectomy.
ALD.10 family.
Boy, aged 6 years, was hospitalized for cervical lymph node
enlargement; he also displayed polyarthritis involving small joints of
the hand and thrombocytopenia, both responsive to steroids.
All the patients displayed anticardiolipin and antinuclear antibodies
(ANA), whereas none of them displayed anti-DNA antibodies. The family
histories for autoimmunity and cancer were collected by interviewing
both parents.
Multiple autoimmune syndrome (MAS) families are those with more than 1 case of organ-specific or systemic autoimmune disorder within the
first- or second-degree relatives.18 All patients attending
the outpatient clinic of the Division of Clinical Immunology and
suffering from connective tissue diseases (CTD) were routinely questioned about family members; a group of consecutive multicase families (2 or more CTD) has been selected for the study. Seventeen subjects with autoimmune manifestations from 7 MAS families were studied. Some members of these families were not included because they
were not available.
MAS.1 family.
Patient MAS.1a is a 55-year-old woman with mixed CTD; MAS1b is a
24-year-old woman with undifferentiated connective tissue disease
(UCTD), niece of MAS1a; MAS.1c is a 45-year-old woman with UCTD, niece
of MAS.1a.
MAS.2 family.
MAS.2a is a 70-year-old woman with Sjögren syndrome (SS); MAS.2b
is a 37-year-old woman with SS, daughter of MAS.2a.
MAS.3 family.
MAS.3a is a 43-year-old woman with UCTD; MAS.3b is a 12-year-old girl
with insulin-dependent diabetes mellitus (IDDM), daughter of MAS.3a;
MAS.3c is a 21-year-old woman with systemic lupus erythematosus (SLE),
sister of MAS.3a (not available).
MAS.4 family.
MAS.4a is a 60-year-old man with SLE; MAS.4b is a 31-year-old man,
healthy but ANA positive, son of MAS.4a; MAS.4c is a 30-year-old woman
with UCTD, daughter of MAS.4a.
MAS.5 family.
MAS.5a is a 34-year-old woman with SS; MAS.5b is a 34-year-old woman
with SS, homozygous twin of MAS.5a (not available); MAS.5c is an
8-year-old boy with autoimmune thrombocytopenia, son of MAS.5b.
MAS.6 family.
MAS.6a is an 11-year-old girl with SLE; MAS.6b, MAS.6c, and MAS.6d were
the mother, the father, and the 14-year-old sister of MAS.6a, and
displayed serum ANA without clinical manifestations. MAS.6d also
displayed high titer of anti-dsDNA and anticardiolipin antibodies.
MAS.7 family.
MAS.7a is a 70-year-old woman with rheumatoid arthritis (RA); MAS.7b is
a 30-year-old man with SLE, son of MAS.7a (not available).
Serologic positivities were reported in the family history and
subsequently confirmed by our clinical pathology laboratory.
Informed consent was obtained from all subjects analyzed or from
their parents.
Immunophenotype analysis
Expression of surface molecules was evaluated by direct
immunofluorescence and cytofluorimetric analysis (FACScan. Becton Dickinson, San Jose, CA). The following mAb were used: anti-CD3 (Leu-4), -CD4 (Leu-3a), -CD8 (Leu-2a), -TCR (Becton Dickinson), and -Fas (Immunotech, Marseilles, France). CD4 and CD8 DN
TCR-positive cells were detected by 2-color immunofluorescence,
using fluorescein isothiocyanate (FITC)-conjugated anti-TCR mAb
and phycoerythrin (PE)-conjugated anti-CD4 and anti-CD8 mAbs. Fas was
detected by 2-color immunofluorescence on resting or activated T cells,
using PE-conjugated anti-CD3 mAb and FITC-conjugated anti-Fas mAb
(Chemicon, Temecula, CA). Nonspecific background fluorescence was
established with the appropriate isotype-matched control mAb (Becton
Dickinson). Antigenic density was expressed as median fluorescence
intensity ratio (MFI-R) of total lymphocytes according to the following formula: MFI-R = MFI of sample histogram (arbitrary units)/MFI of
control histogram (arbitrary units).
Cell death assay
Cell death induced by Fas or C2-ceramide was evaluated as previously
reported10 on T-cell lines obtained by activating
peripheral blood mononuclear cells (PBMC) with phytohemagglutinin (PHA)
at days 0 (1 µg/mL) and 15 (0.2 µg/mL) and cultured in RPMI
1640 + 10% FCS + recombinant IL-2 (5 U/mL) (Biogen, Geneva,
Switzerland). Fas function was assessed 6 days after the second
stimulation (day-21 T cells). In some experiments Fas function was also
evaluated 6 days after the first stimulation (day-6 T cells). Cells
were incubated with control medium or anti-Fas mAb (CH11, IgM isotype) (1 µg/mL) (UBI, Lake Placid, NY) in the presence of recombinant interleukin 2 (rIL-2) (5 U/mL) to minimize spontaneous cell death. Cell
survival was evaluated after 18 hours by counting live cells in each
well by the trypan blue exclusion test. The same conditions were used
to measure cell death induced by methyl-prednisolone (100 µmol/L)
(PDN) (Upjohn, Puurs, Belgium) or C2-ceramide (50 µmol/L)
(N-acetyl-D-sphingosine) (Sigma, St Louis, MO).
Assays were performed in triplicate and analyzed by a blind observer. Cells from 2 normal donors were included in each experiment as a
positive control. Results were expressed as relative cell survival percentage, calculated as follows: (total live cell count in the assay
well/total live cell count in the control well) × 100.
Spontaneous cell loss in the control well was always less than 10% of
the seeded cells and similar in cultures from the patients and normal donors. This protocol was chosen in preliminary experiments, when several anti-Fas mAb concentrations (10, 1, 0.1 µg/mL) and incubation times (1, 4, 8, 18, 48, 72 hours) were used to induce cell death in
T-cell lines cultured for 3, 6, 9, 15, 18, 21, and 24 days with
PHA+IL-2. Cell death was evaluated both indirectly, by counting total
surviving cells by the trypan blue exclusion test, or directly, by FACS
determination of the proportion displaying shrunken/hypergranular morphology or those displaying DNA fragmentation after staining with
propidium iodide or those stained by annexin V. The protocol chosen was
found to give the most reproducible results. It evaluates the overall
cell survival at each time point and was more sensitive than the other
techniques detecting the instantaneous proportion of dying cells at
each time. Fas-induced cell death was always less striking in these
polyclonal T-cell lines than in stabilized tumor cell lines, because it
was slower and more asynchronous.10
The normal range of responses of day-21 T cells to Fas-, ceramide- and
PDN-induced T-cell survival, defined as the mean ± 2 SD of data
obtained from 75 normal donors, were 60 ± 22 (Fas), 58 ± 32
(ceramide), and 49 ± 26 (PDN), respectively (relative cell
survival percentage).
Evaluation of cell staining with annexin V was performed using the
Annexin-V-Fluos kit (Boehringer Mannhein, Gmbh, Germany). Briefly,
106 cells were stained with annexin-V 1:50 and propidium
iodide (1 µg/mL) in 10 mmol/L Hepes/NaOH pH7.4, 140 mmol/L NaCl, 5 mmol/L CaCl2 and analyzed by flow cytometry gating on
propidium iodide-negative cells.
Analysis of the Fas gene
Mutation analysis of the Fas gene was performed by
single-strand conformation polymorphism (SSCP) and cDNA sequencing, as previously reported.10 Total RNA was extracted from fresh
PBMC with the Ultraspec kit (Biotex, Houston, TX). Total RNA (2 µg) was used as a template for cDNA synthesis with the Promega cDNA synthesis kit. The entire coding sequence of the Fas gene was amplified in a single 1114 bp segment and in overlapping 203-302 bp
segments. Polymerase chain reaction (PCR) was performed with 25 pmol of
each primer and one fourth of the cDNA synthesis reaction (35 cycles
per fragment) and using the primers previously reported.10 SSCP analysis was performed for each PCR product using 32P
labeling and a protocol previously reported.10 Sequencing of cDNA was performed with a dye terminator DNA sequencing kit (Applied
Biosystems, Perkin-Elmer, Foster City, CA) on an ABI Model 373A
automated DNA sequencer (Applied Biosystems, Perkin-Elmer). Each DNA
fragment was sequenced twice using primers from both ends and PCR
products from 2 independent PCR reactions. Only patients with ALD were
analyzed for mutations.
Statistical analysis
Cancer frequency was analyzed by comparing the observed cases of
cancer in patients' families with expected cases based on site, age
and calendar period, regional-specific incidence, and mortality rates
multiplied by observed number of person-time at risk. Person-time of
grandparents cumulated only starting from the year of birth of father
(or mother) who generated the sick child; person-time of father (or
mother) cumulated only from the year of birth of the sick child.
Observed and expected cases were compared by means of standardized
mortality (SMR) and standardized incidence (SIR) ratios and their 95%
confidence intervals (CI).19 To compare frequencies of
autoimmune subjects the chi-square with Yates correction or Fisher
exact tests were used. Control families were obtained from 21 consecutive children observed in the general practitioner outpatient
clinic. They were interviewed using the same criteria and by the same
person who interviewed the families of the patients with ALD.
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Results |
Deficiency of the Fas cell death pathway in families of ALD
patients
Figure 1 shows the death responses to anti-Fas mAb of T-cell lines from
10 ALD patients. All patients were resistant to Fas-induced cell death.
The defect was displayed by both day-6 and day-21 cultures. Moreover,
it was detected by evaluation of both cell survival by the trypan blue
exclusion test and cell apoptosis by annexin V staining. However, cell
survival on day-21 T cells detected the maximal difference between
patients and normal donors. Fas function was not abolished because cell
death increased when incubation was prolonged to 48 hours or Fas
triggering was potentiated by the anti-IgM serum (reference 10 and data
not shown). Search for DN T-cell expansion in fresh PBMC detected
expansion only in patient 9 (21% DN T cells), whereas DN cells were
less than 1% in the other patients. In line with our previous
findings, lymphocyte subset distribution and proliferative response to
mitogens were normal. Expression of CD38, HLA-DR, and Fas was slightly increased, which indicated presence of activated cells and was in line
with what is observed in other autoimune diseases (reference 10 and
data not shown). Search for mutations of the Fas gene by SSCP
analysis and sequencing of the entire coding region did not detect any
causal mutation (data not shown).
To assess the inherited component of the Fas apoptosis pathway
deficiency, we evaluated cell death induction in T-cell lines derived
from both parents. Moreover, we analyzed 4 close relatives (3 siblings
and 1 aunt) who were available. All these subjects were healthy except
the aunt of patient 5, who displayed chronic splenomegaly,
hypergammaglobulinemia, and autoimmune thrombocytopenia. We also
evaluated the response to ceramide, whose pathway partially overlaps
that of Fas,20,21 and to PDN, which does not involve the
Fas system. As shown in Figure 2, 9 of 10 mothers and 8 of 10 fathers were resistant to Fas-induced cell death.
Moreover, the 3 parents with normal response (ie, fathers 1 and 3, and
mother 10) were near the upper limit of the normal range. Seven
patients and 8 parents (4 of 10 mothers and 4 of 10 fathers) were also resistant to ceramide. Father 3 and mother 10 also displayed a normal
response to ceramide, whereas father 1 was resistant to ceramide. All 4 relatives were resistant to either Fas- or ceramide-induced cell death;
2 were resistant to both Fas and ceramide, 1 to Fas only, and 1 to
ceramide only. By contrast, all subjects displayed a normal cell death
response to PDN. Similar data were obtained by annexin V staining (data
not shown).
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| Fig 2.
Fas-, ceramide-, and PDN-induced T-cell death in 10 ALD
families.
Members of each family are marked with the indicated symbols and were
evaluated in the same experiment (different from that shown in Figure
1). Two or 3 normal controls were included in each experiment and are
shown in lane C. Day-21 T cells were treated with the indicated reagent
and survival was assessed after 18 hours. Results are expressed as
specific cell survival percentage. The horizontal lines indicate the
upper limit of the normal range calculated as the mean + 2 SD from data obtained from 75 normal donors. Among the
close relatives, the aunt was selected because she displayed an
ALPS-like clinical pattern, whereas the 3 healthy siblings were
casually selected. All relatives analyzed are included in the text. C
indicates normal controls; F, fathers; M, mothers; Pt, ALD patients; R,
close relatives.
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Statistical analysis showed that frequencies of resistance to Fas and
ceramide were significantly higher in families of ALD patients than in
normal controls. By contrast, frequency of resistance to PDN was not
significantly increased (Table 1). Search
for DN T-cell expansion in fresh PBMC did not detect expansion in any
parent or close relative. Moreover, lymphocyte subset distribution and
expression of CD38, HLA-DR, and Fas were always normal.
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Table 1.
Frequency of Fas-, ceramide-, and PDN-resistant subjects
in normal controls and different patient groups
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Fas expression was evaluated in each T-cell line by direct
immunofluorescence on the same day in which the cell death assay was
performed and was always in the normal range; moreover, search for DN T
cells in fresh PBMC by 2-color immunofluorescence did not reveal
expansion of these cells in any of these subjects (data not shown).
Increased frequency of autoimmune diseases and cancer in the
families of ALD patients
The decreased Fas- or ceramide-induced cell death displayed by most
members of the patients' families supports the possibility that ALD
has a genetic component. Because decreased Fas function may favor
development of autoimmunity and cancer, we determined whether frequency
of these diseases was increased in these families. Family histories
were collected up to patients' grandparents and pedigrees are shown in
Figure 3.
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| Fig 3.
Pedigrees of the ALD families.
Black symbols mark subjects with cancer. Arabic numbers indicate the
type of cancer: 1, gastric carcinoma; 2, lymphoma; 2B, Hodgkin
lymphoma; 3, small cell lung cancer; 4, polycythemia vera; 5, colon
carcinoma; 6, breast carcinoma; 7, lung carcinoma; 8, kidney
adenocarcinoma; 9, squamous head carcinoma. Gray symbols mark subjects
with autoimmune disease. In each family the ALD patient is the single
affected individual in the third generation. Lower-case letters
indicate the type of autoimmune disease: a, IDDM; b, chronic hemolytic
anemia, c, autoimmune thrombocytopenia; d, neutropenia; e, SLE; f,
multiple sclerosis; g, RA; h, autoimmune hepatitis; i, ankylosing
spondylitis; j, polyarthritis.
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Analysis of autoimmune disease was performed by comparing frequencies
of autoimmune diseases in the patients' families and in those of 21 consecutive children observed in the general practitioner outpatient
clinic (Table 2) (control families). We
found that families of ALD patients displayed a significantly higher
frequency of autoimmune diseases than control families (9.3% versus
2.6%, P = .014). The difference was mostly due to males
(11.1% versus 0.8%, P = .0035), because frequency of
autoimmune diseases in females was similar in the 2 groups (7.4%
versus 4.5%). To investigate the inheritance pattern, we evaluated
frequency of autoimmunity in paternal and maternal lineages. In the
maternal lineage, no difference was found between ALD patients and
controls (1.9% versus 3.9%), whereas in the paternal lineage,
frequency of autoimmune diseases was significantly higher in the
families of ALD patients (19.6% versus 1.7%, P = .0002).
The significantly increased frequency was displayed by both males
(18.8% versus 1.4%, P = .0036) and females (21.4% versus
2.1%, P = .035).
Cancer frequency was analyzed by comparing the observed cases of cancer
in these families with the frequency expected from the mortality rates
for our region (ie, Piedmont, Italy) because official registers were
available (Table 3). In the maternal family
line, ALD patients displayed significantly higher incidence (SIR = 1.87; CI, 0.99-3.2) and mortality (SMR = 2.75; CI,
1.26-5.23) for cancer than expected. The significantly increased
frequency was displayed by females (SIR = 2.68; CI, 1.15-5.27;
SMR = 4.63; CI, 1.49-10.83) and not by males. No increased frequency
was found in the paternal family line. These data support the
possibility that genetic alterations of the Fas signaling pathway may
be a novel genetic factor predisposing to cancer development and
suggest that the trait is controlled by the maternal line.
To confirm these data, we compared SIR and SMR for cancer in families
of ALD patients and in the control families and calculated the
corresponding ratios. Moreover, we applied the Kaplan-Meier method with
the log-rank test. Both analyses gave results consistent with those
shown in Table 3 (data not shown).
Deficiency of Fas-induced cell death in families with high frequency
of autoimmunity
To further assess the possibility that deficiency of Fas-induced
cell death may predispose to development of autoimmune diseases different from typical ALD, we evaluated Fas- and ceramide-induced cell
death in T cells from 17 patients displaying autoimmune diseases from 7 families with a high frequency of autoimmunity. In each family, several
cases of different autoimmune diseases were diagnosed, with the
exception of the MAS.6 family, which displayed 1 subject with frank
autoimmunity and 3 first-degree relatives with high amounts of serum
autoantibodies in the absence of overt autoimmune manifestations. We
found that 8 of 17 subjects were resistant to Fas-induced cell death
and 2 of 17 were resistant to ceramide; 7 were resistant to Fas only, 1 was resistant to ceramide only, and 1 was resistant to both Fas and
ceramide; 2 patients were also resistant to PDN (Figure
4). Similar data were obtained by evaluating cell death by annexin V staining (data not shown). Statistical analysis showed that frequency of resistance to Fas was
significantly higher in families of ALD patients than in normal controls. By contrast, frequencies of resistance to ceramide and PDN
were not significantly increased (Table 1). Fas expression in all
T-cell lines was always in the normal range, and no expansion of DN T
cells was detected in PBMC (data not shown).
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| Fig 4.
Fas-, ceramide-, and PDN-induced T-cell death in
autoimmune subjects from 7 MAS families.
Black symbols mark subjects with autoimmune disease and gray symbols
subjects with serologic autoimmunity without overt disease. Results are
expressed as in Figure 1. Two or 3 normal controls were included in
each experiment and are shown in Table 1.
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Discussion |
Fas function defects due to inherited loss-of-function mutations of
the Fas gene are responsible for the pattern of
autoimmunity/lymphoproliferation displayed by patients with ALPS and
lpr mice.1-11 Both the human and mouse
immunopathologic patterns are characterized by peripheral expansion of
DN T cells. Interestingly, most ALPS patients are heterozygous for Fas
mutations, but their parents who are heterozygous for the mutation are
generally healthy. Therefore, it has been suggested that heterozygous
symptomatic ALPS patients inherit a single mutation in the Fas
gene from 1 parent and a second abnormality, not directly involving the
Fas gene, from the other. We have recently reported altered
Fas-induced cell death in patients displaying an ALPS-like clinical
pattern in the absence of Fas mutation and peripheral expansion of DN T
cells. The ALD abnormality seems to involve the Fas signaling pathway
downstream from Fas. This possibility has been recently confirmed in 2 patients with an ALPS-like clinical pattern who displayed mutations of
caspase-10 in the absence of mutations of Fas and FasL.22 A
recent classification uses the terms ALPS type Ia and Ib for the
disease with mutations of Fas and FasL, respectively, and ALPS type II
for the disease without such mutations.23 Our patients
differ from the ALPS type II patients reported by Wang et
al22 because only one of them displayed DN
cell expansion. Our data did not rule out the possibility that the Fas
defect was the outcome of a lymphocyte functional state induced by
unknown environmental factors. However, the pediatric onset,
the similarities with ALPS, the consanguineous parents of 1 patient,
and the family history with autoimmunity of some patients favored a
genetic component. Our present finding that 17 of 20 parents of the ALD
patients display defective Fas-induced cell death strongly supports
this model. The fact that most were healthy suggests that ALD patients
inherited 2 mutations of the Fas signaling pathway and that presence of
both mutations is required for ALD expression. Interestingly, we
detected 3 patterns of cell death deficiency. Most subjects displayed
dual resistance to Fas- and ceramide-induced cell death, but some were
resistant to Fas and not to ceramide, or vice versa. These data suggest
that different subjects carry different genetic alterations hitting
different levels of the Fas signaling pathway and that ALD expression
requires accumulation of several hits in the same subject. Moreover,
these data suggest that the death pathways triggered by Fas and
ceramide are in part distinct. The current opinion is that Fas
triggering activates a caspase cascade. Hydrolysis of the phospholipid
sphingomyelin is an alternative pathway that seems to boost the caspase
pathway, because it is triggered by caspases and potentiates caspase
activation.20,21 However, the role played by ceramide
production in Fas signaling is debated.24,25 Therefore, it
is noteworthy that some patients were resistant to ceramide, but
responded normally to Fas triggering, because this shows that Fas can
also function normally even when the ceramide pathway is nonfunctional.
Autoimmune diseases displayed by patients with ALPS and ALD are
generally hematologic autoimmunities, that is, autoimmune anemia, thrombocytopenia, or neutropenia. Patients with ALPS, but not
ALD, may also display vasculitis. Moreover, 1 ALPS patient reported by
Pensati et al displayed autoimmune hepatitis.7 We now
report 2 lines of evidence showing that decreased function of Fas may
also predispose to development of autoimmune patterns different from
the rare ALPS/ALD pattern. The former line of evidence was suggested by
the higher frequency of autoimmune diseases in families of ALD patients
than in control families. Interestingly, this increased frequency was
selectively displayed by the paternal lineage. The latter line of
evidence was obtained by showing that resistance to Fas-induced cell
death was detectable in a substantial proportion of autoimmune
patients belonging to families with a high frequency of autoimmune diseases.
Interestingly, the pattern of autoimmune diseases displayed by these
patients and by families of ALD patients was heterogeneous, because it
comprised multiple sclerosis, IDDM, SS, RA, SLE, autoimmune hepatitis,
and ankylosing spondylitis.
The possibility that genetically based deficiencies of Fas may
predispose to development of organ-specific cell-mediated autoimmune diseases, such as IDDM and multiple sclerosis, apparently contradicts the model suggested by several authors that development of these diseases involves cytotoxic mechanisms mediated by the Fas/FasL system.
This model was suggested by the observation that high levels of Fas and
FasL are expressed in pancreatic islet, thyroid, and central nervous
tissue in IDDM, thyroiditis, and multiple sclerosis,
respectively.26 Moreover, the lpr character seems to protect animals from development of autoimmunity in experimental models of IDDM and multiple sclerosis.27-29 However, it
must be emphasized that we detected decreased Fas function in selected patients, which does not imply that decreased Fas function is a common
cause of autoimmunity. In line with this possibility, Fas function has
been reported to be increased in patients with SLE,30,31
and we did not detect defective Fas function in random patients with
autoimmune thyroiditis (n = 19) or IDDM (n = 13) (unpublished
data). Moreover, predisposition to autoimmune diseases is
multifactorial and may involve factors controlling autoantigen expression, lymphocyte responsiveness, and immune effector functions. Therefore, decreased function of the Fas system may protect from development of autoimmunity in certain genetic contexts and models of
autoimmunity, but may be detrimental in others. Moreover, the expression pattern of the disease may be influenced by residual Fas
function and function of other systems involved in apoptosis induction,
such as the TNF and TRAIL systems, which use signaling pathways partially overlapping that of Fas.1,2,32,33
Our data also show that the ALD genetic trait also predisposes to
development of cancer. The ALD families displayed several types
of cancers without apparent predominance of any particular type. They
did not display predominance of lymphoid neoplasia, as might have been
expected from anecdotal report of lymphoma development in families of
ALPS patients. However, immune alterations are partly different in ALD
and ALPS patients, as shown by the expansion of DN cells in ALPS only,
and these differences might influence development of lymphoid
neoplasia. Interestingly, increased predisposition to development of
cancer was inherited only via the maternal lineage and was significant
in females and not in males. Moreover, analysis of each pedigree
suggests that ALD families are heterogeneous with respect to
predisposition to cancer and autoimmunity, because only families 5 and
7 displayed several cases of both autoimmune diseases and cancers,
whereas families 1, 8, 9, and 10 displayed several cases of autoimmune
disease only, and families 3, 4, and 6 several cases of cancers only. Interestingly, pedigrees of families 1, 3, 4, 5, 6, and 7 showed an
inheritance pattern compatible with X-linked inheritance, assuming that
ALD and tumors are manifestations of the same disease. These data
support the possibility that genetic alterations of the Fas signaling
pathway may be a novel genetic trait predisposing to cancer development
and suggest that it is controlled by a gene on the X chromosome.
In conclusion, our data suggest that ALD development requires
accumulation of several genetic defects hitting cell apoptosis in the
same subjects. Some of these defects are also associated with increased
development of common autoimmune diseases, others to increased
development of cancer. These increased predispositions seem to be
inherited independently because they are differently displayed by ALD
families and inherited through different family lines.
| |
Footnotes |
Submitted April 28, 1999; accepted January 17, 2000.
Supported by Telethon grant No E566 (Rome), Associazione Italiana
Ricerca sul Cancro (A.I.R.C., Milan), MURST ex-40% (Rome), AIDS
Project (Istituto Superiore di Sanità, Rome). S.B. was supported by Lega Italiana Lotta contro i Tumori (Novara).
U.R. and S.B. contributed equally to this work.
Reprints: Umberto Dianzani, Dipartimento di Scienze
Mediche, Via Solaroli, 17, I-28100 Novara, Italy; e-mail:
dianzani{at}med.no.unipmn.it.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction