By
From the * Division of Immunogenetics, Department of Pediatrics, University of Pittsburgh School of
Medicine, Rangos Research Center, Children's Hospital of Pittsburgh, Pittsburgh, PA 15213; Department of Experimental Medicine and Biochemical Sciences, University of Rome "Tor Vergata",
00133 Rome, Italy; § Department of Pathology, University of Pittsburgh School of Medicine,
Pittsburgh, PA 15213; and
Laboratory of Immunology, Endocrinology Section, Institute of Clinical
Medicine, University of Palermo, 90127 Palermo, Italy
Fas is an apoptosis-inducing surface receptor involved in controlling tissue homeostasis and
function at multiple sites. Here we show that cells from the pancreata of newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients express Fas and show extensive apoptosis
among those cells located in proximity to Fas ligand-expressing T lymphocytes infiltrating the
IDDM islets. Normal human pancreatic
cells that do not constitutively express Fas, become
strongly Fas positive after interleuken (IL)-1
exposure, and are then susceptible to Fas-mediated apoptosis. NG-monomethyl-L-arginine, an inhibitor of nitric oxide (NO) synthase, prevents
IL-1
-induced Fas expression, whereas the NO donors sodium nitroprusside and nitric oxide
releasing compound (NOC)-18, induce functional Fas expression in normal pancreatic
cells.
These findings suggest that NO-mediated upregulation of Fas contributes to pancreatic
cell
damage in IDDM.
The interaction of Fas (CD95/Apo-1) with its ligand
promotes the deletion of potentially harmful, damaged, or unnecessary cells during the immune response.
This interaction also regulates tissue remodelling and homeostasis. Impaired Fas-induced apoptosis results in abnormal cell proliferation and accumulation, whereas inappropriate expression or excessive Fas activity causes tissue damage (1).
Insulin-dependent diabetes mellitus (IDDM)1 is a T cell-
mediated autoimmune disease resulting from a selective destruction of pancreatic Normal pancreatic Subjects.
The IDDM pancreata were from two boys (ages 12 and
11), who died in 1992 (patient No. 1) and 1993 (patient No. 2),
respectively, after a diagnosis of type I diabetes mellitus complicated by diabetic ketoacidosis (14). Despite appropriate treatment
based on fluid rehydration combined with insulin infusion therapy, both children developed severe brain edema with cerebellar
tonsillar herniation. Upon admission to Children's Hospital of
Pittsburgh (Pittsburgh, PA), they were found to be brain dead.
After obtaining an informed consent from the parents, the tail,
body, and part of the head of the pancreas were obtained at autopsy. In both cases, molecular HLA typing revealed heterozygosity at both DQA1 (i.e., patient No. 1: *0501, *0301; and patient No. 2: *0101, *0501) and DQB1 (i.e., patient No. 1: *0301,
*0302; and patient No. 2: *0201, *0501) loci. Thus, both patients
had the possibility of forming "diabetogenic" ( Flow Cytometry Analysis of Islet Cells.
Pancreatic islets were isolated by the intraductal pancreatic distension method as previously described (8). Single cell suspensions were obtained by dissociating islets with 3 mM EGTA, 20 mg/ml BSA, 2.8 mM
glucose, and 0.83 µg/ml trypsin (8). Human IFN- Apoptosis Detection of Islet Cells.
After a 24-h culture in the
presence of 50 U/ml IL-1 Immunostaining Procedure.
Pancreas fragments (0.5 cm) were
snap frozen in isopentane, chilled at FasL Messenger RNA Analysis.
Total cytoplasmic RNA was
prepared from the NHP-purified islets, IDDM-purified islets, and
resting and PMA/ionomycin-activated PBLs from healthy individuals. The RNA was extracted using TRIZOL reagent
(GIBCO BRL, Gaithersburg, MD) according to the manufacturer's instructions. For each preparation, 3 µg of total RNA was
reverse transcribed into a single-stranded cDNA (GeneAmp
RNA PCR Kit; Perkin-Elmer, Roche Molecular Systems, Inc.,
Branchburg, NJ).
In Situ Apoptosis Studies.
Pancreas cryostat sections (5 µm)
were mounted onto microscope slides and fixed with 4% paraformaldehyde. Permeabilization of the tissue was determined by incubation with 0.1% Triton X-100, 0.1% sodium citrate (16). The
labeling of 3 To determine whether IL-1
NO is a reactive nitrogen intermediate with multiple biological effects (17). Different from
TNF-
To determine whether Fas is functional and able to induce
a death signal in
To investigate whether Fas is expressed in pancreatic
Fas requires the interaction with its ligand to
transduce the death signal. We therefore searched for FasL
expression in normal and IDDM pancreata. Different from
thyroid follicular cells (20), both the endocrine and the
exocrine cells from NHP were FasL negative, as were the
islet cells from IDDM patients (Fig. 5, A and B). However,
activated T cells infiltrating the islets were strongly FasL
positive (Fig. 5 A), as shown by the colocalization of FasL and CD3 positivity in serial sections (Fig. 4 and 5 A).
Moreover, FasL transcripts were not detected in cDNA
prepared from purified islets of NHP, but were found in
cDNA preparations from diabetic pancreata (Fig. 5 B). Finally, TUNEL analysis of IDDM pancreas specimens
showed extensive apoptosis among Fas-positive
Fas-mediated cytotoxicity can be
induced by both CD4- and CD8-positive T cells, being
the major cytotoxic effector mechanism of CD4 cells (23,
24), which constitute the dominant subset of T cells infiltrating the IDDM pancreata we have examined (14). Multiple islet antigens have been claimed as major targets of the
autoimmune aggression in IDDM, as both humoral and
cell-mediated autoimmune responses are polyclonal and
not During the insulitis process, NO production is probably
not confined to IL-1 cells (4). Two complementary
lytic pathways mediate T cell cytotoxicity: the exocytosis
of perforin-containing granules on cognate target cells, and
the engagement of Fas on cognate or neighboring target
cells by membrane-bound or released Fas ligand (FasL; 5, 6).
cells do not express Fas (7). However, we have recently shown that the exposure of human
cells to IL-1
induces Fas expression on these cells, suggesting the existence of an additional pathway that promotes
cell destruction (8). The possible role of FasL-based cytotoxicity in the pathogenesis of autoimmune diabetes is suggested by recent findings showing protection
from both spontaneous and T cell-transferred diabetes in
Fas negative NODlpr/lpr mice (9). IL-1
has been reported
to be abundantly present during the insulitis process and selectively toxic for pancreatic
cells, as its secretion by activated macrophages induces transcription and translation of
inducible nitric oxide synthase (iNOS) and nitric oxide
(NO) production (10, 11). During the insulitis process,
iNOS is highly expressed in
cells and islet-infiltrating macrophages (12). Although IL-1
-mediated NO production may result in
cell damage, and islet-infiltrating macrophages are a major source of NO, it seems unlikely that
this direct cytotoxic effect of NO is responsible for massive
and selective
cell destruction (13). We therefore studied
the requirement for functional Fas expression in pancreatic
cells, and investigated the possible involvement of Fas
and FasL in the pathogenesis of human IDDM.
Arg-52 and
-non-Asp57) DQ heterodimers (15). Islet cell antibodies were
>20 Juvenile Diabetes Foundation (JDF) units in the serum of
patient No. 1, and <20 in the serum of patient No. 2 (14). 10 normal human pancreas (NHP) specimens were provided by the
Consorzio Centro Sud Trapianti (Palermo, Italy) and the Thomas
E. Starzl Transplantation Center (Pittsburgh, PA) under their respective Institutional Review Board approvals. They were obtained from three female and seven male nondiabetic cadaveric donors (mean age was 25 yr old).
(specific
activity of 106 U/mg; Sigma Chemical Co., St. Louis, MO),
TNF-
(specific activity of >2 × 107 U/mg; Boehringer Mannheim, Indianapolis, IN), IL-1
(specific activity of ~5 × 108 U/
mg; Genzyme, Cambridge, MA), sodium nitroprusside (SNP),
NG-monomethinyl-L-arginine (L-NMMA), and NG-monomethyl-
D-arginine (D-NMMA; Sigma Chemical Co.), and NO releasing compound (Z)-1-[2-(2-aminoethyl)-(N)-(2-ammonioethyl)-
ammo]diazen-1-ium-1,2-diolate] (NOC-18), L-arginine, and
D-arginine (Alexis Corp., Laufelfingen, Switzerland) were added
to the cultures at time 0. Cells were maintained in culture for 72 h
and harvested for evaluation of Fas expression at different time
points. Islet cells were first permeabilized in a fixative solution
containing 0.25% paraformaldehyde and 0.2% Tween 20 for the
labeling with anti-insulin mAb (IgG2a; Novo Nordisk, Bagsvaerd, Denmark), revealed by an FITC-conjugated goat anti-
mouse Ig antibody. Cells were then incubated for 10 min with
6% normal mouse serum, treated with phycoerythrin-conjugated anti-CD95 (DX2, IgG1; PharMingen, San Diego, CA), washed
twice in cold PBS/azide, and resuspended at 106 cells/ml for
cytofluorometric analysis. An isotype-matched antibody was used
as a negative control. Relative fluorescent intensities of individual
cells were analyzed using a FACScan® flow cytometer (Becton
Dickinson, Mountain View, CA).
, 0.5 mM SNP, or IL-1
plus L-NMMA,
dispersed islet cells were incubated with an isotype-matched control mAb or with 200 ng/ml of anti-Fas mAb (IgM, clone CH-11;
Upstate Biotechnology Incorporated, New York, NY), or 10 µg/ml of anti-Fas-blocking mAb (IgG1, ZB4; Kamiya Biomedical Company, Tukwila, WA) for a total of 48 h. The percentage of cells undergoing apoptosis was measured by hypotonic fluorochrome solution staining (50 µg/ml propidium iodide in 0.1%
sodium citrate plus 0.1% Triton X-100; Sigma Chemical Co.)
detected with a FACScan® flow cytometer. Cell recovery was
determined by evaluating the number of surviving insulin or glucagon positive cells recognized by specific mAbs (Novo Nordisk)
out of 500 cells counted, revealed by a FITC-conjugated goat
anti-mouse Ig antibody and observed by fluorescence microscopy.
150°C and kept at
80°C
until used. Serial cryostat pancreatic sections (5 µm) were allowed
to equilibrate to room temperature and exposed to absolute acetone for 10 min before starting the alkaline phosphatase anti-alkaline phosphatase (APAAP) complex procedure, or to a hydrogen
peroxide methanol solution for double staining (Dako Double
Stain Kit; Dako Corp., Santa Barbara, CA). Detection of antiglucagon antibody (Dako Corp.) binding was accomplished using a
horseradish peroxidase-rabbit anti-horseradish peroxidase complex (Dako Corp.) and revealed by 3,3
-diamino benzidine tetrahydrochloride. Bound anti-CD3 (Dako Corp.), anti-CD14
(Dako Corp.), anti-human perforin (provided by E.R. Podack,
University of Miami, Miami, FL), anti-CD95 (DX2, IgG1;
PharMingen), and anti-FasL (NOK-1, IgG1; PharMingen) mAbs were detected with APAAP mouse (Dako Corp.) and revealed by
a Fast Red colorimetric substrate. Hematoxylin was used as a
counterstain. Control tissue sections were subjected to identical
treatment and analysis using irrelevant isotype-matched mAbs.
-OH fragmented DNA ends (TdT-mediated
dUTP-X nick end labeling; TUNEL) was carried out by an in
situ apoptosis detection kit (In Situ Cell Death Detection, AP;
Boehringer Mannheim) following the instructions of the manufacturer. Detection of labeled ends was done with an antifluorescein antibody (Fab
2 fragment) conjugated with alkaline phosphatase. 5-bromo-4-chloro-3-indolylphosphate (Dako Corp.) was
used as substrate. Sections were then counterstained with hematoxylin.
IL-1 Induces Selective Fas Expression in
Cells.
selectively induces Fas expression in
cells but not in other pancreatic endocrine cells, normal dispersed islet cells were treated 24 h with 50 U/ml IL-1
,
double stained with anti-insulin and anti-Fas mAbs, and
analyzed by flow cytometry. Fig. 1, A and B shows that
IL-1
-induced Fas expression in islet cells was restricted to
insulin-producing cells. The induction of Fas expression in
cells peaked after ~24 h of IL-1
stimulation, and was not
observed in any cell type after exposure to TNF-
or IFN-
,
alone or in combination (Fig. 1, C and D). These data suggest that some molecular component generated by IL-1
stimulation may be responsible for Fas induction in
cells.
Fig. 1.
Fas expression induced by IL-1 on enzymatically dispersed NHP islet cells.
Control (A) or Fas (B) expression
on insulin-negative and -positive
islet cells exposed for 24 h to 50 U/ml IL-1
. (C) Fas expression
on
cells exposed for 24 h to
varying (i.e., ranging from 25 to
1,000 U/ml) doses of recombinant IL-1
(closed squares), TNF-
(open diamonds), IFN-
(closed diamonds), TNF-
plus IFN-
(open squares), or tissue culture
medium alone (open circle). (D)
Time-course experiment (one
of eight performed on eight different human pancreata) demonstrating Fas expression in
cells
incubated with IL-1
(50 U/
ml), TNF-
, IFN-
, and IFN-
plus TNF-
(500 U/ml).
[View Larger Version of this Image (28K GIF file)]
Cells and Mediates IL-1
-
induced Fas Upregulation.
or IFN-
, IL-1
is a potent inducer of iNOS in islet cells (11). To investigate the possible contribution of
NO in IL-1
-induced Fas expression, we treated normal
dispersed islet cells with IL-1
in the presence or absence
of the nitric oxide synthase inhibitor L-NMMA. Targeting NOS activation by L-NMMA but not D-NMMA (18),
treatment inhibited IL-1
-induced Fas expression (Fig. 2,
A and B). Also, inhibition by L-NMMA was completely
reversed by excess of L-arginine, but not by D-arginine
(19), suggesting that NO generation is directly responsible
for IL-1
-induced Fas upregulation. To confirm this hypothesis, we exposed islet cells to the NO donors SNP and
NOC-18 for 24 h and examined them for Fas expression.
Both SNP and NOC-18 were extremely effective in inducing Fas expression in
cells (Fig. 2 A). As expected,
L-NMMA did not interfere with NO-induced (NOC-18)
Fas expression (Fig. 2 B). Taken together, these results suggest a primary role for NO in sensitizing
cells to Fas-induced apoptosis.
Fig. 2.
Cytofluorometric analysis of enzymatically dispersed islet
cells from NHPs. (A) Insulin-positive cells were gated for subsequent analyses. Untreated cells are Fas negative (control). Treatment with 50 U/
ml IL-1 or 0.5 mmol/liter SNP or 0.5 mg/ml NOC-18, induces Fas expression on gated insulin-positive cells. Exposure to 3 mmol/liter L-NMMA
suppresses IL-1
-induced Fas on
cells. Reactivity with irrelevant isotype-matched mAbs is shown in gray. (B) Mean values of two experiments confirming results shown in A and demonstrating that exposure to
3 mmol/liter D-NMMA was not able to antagonize IL-1
-mediated Fas
expression on
cells. The addition of L-arginine (15 mM) overrode
L-NMMA inhibitory effect on IL-1
-induced Fas, whereas D-arginine
(15 mM) did not. Also, L-NMMA did not reverse NOC-18-promoted
Fas upregulation.
[View Larger Versions of these Images (27 + 14K GIF file)]
Cells for Fas-mediated Destruction.
cells, we incubated IL-1
- or SNP-
treated and untreated islet cells with an agonist (CH-11) or
a blocking anti-Fas (ZB4) mAb and examined them for evidence of apoptosis. Treatment with CH-11 mAb in the
absence of IL-1
or SNP did not trigger apoptosis. By contrast, incubation with CH-11 dramatically increased apoptosis in SNP- or IL-1
-treated-cells (Fig. 3 A). Immunofluorescence staining and fluorescence microscopy analysis
revealed that after 24 h, essentially all cells surviving IL-1
and anti-Fas treatment were glucagon-positive
cells,
whereas the recovery of insulin-producing cells was extremely low, indicating that only
cells underwent apoptosis (Fig. 3 B). Moreover, L-NMMA was able to completely suppress IL-1
-induced apoptotic cell death,
whereas the anti-Fas antagonist ZB4 did not interfere with
IL-1
- or SNP-induced apoptosis (Fig. 3 A). Thus, NO
mediates both Fas-independent and Fas-dependent
cell
apoptosis induced by IL-1
.
Fig. 3.
Induction of apoptosis in human pancreatic islet cells by anti-Fas mAb (CH-11). (A) Islet cells incubated for 24 h in tissue culture medium and then exposed for an additional 24 h to 200 ng/ml CH-11 mAb,
or in medium supplemented with IL-1 (50 U/ml) or 0.5 mmol/liter
SNP followed by control IgM, or in medium containing IL-1
or SNP
followed by 10 µg/ml ZB4 or CH-11 mAb, or in medium containing
IL-1
plus 3 mmol/liter L-NMMA followed by CH-11 mAb, were processed for DNA content analysis by staining. Percentages of apoptotic
(i.e., hypodiploid) nuclei are indicated between shift markers, plotted on
log histograms as red fluorescence intensity (x-axis) versus relative nuclei
number (y-axis). (B) Evaluation of cell survival of human
(left) and
cells (right) cultured in culture medium for 24 h and then for 24 h in medium in the absence of IL-1
, in medium supplemented with IL-1
(50 U/ml; open squares), and control mAb (open circles), or in medium with IL-1
and CH-11 mAb (closed circles), or in medium with IL-1
plus L-NMMA
and CH-11 mAb (closed triangles). Data shown represent the values at the
indicated time points of a representative experiment out of a total of seven
independent experiments performed.
[View Larger Version of this Image (28K GIF file)]
Cells During Insulitis.
cells during the insulitis process, we next analyzed expression and distribution of Fas in endocrine and infiltrating cells in pancreatic specimens from newly diagnosed IDDM patients.
Immunohistochemical analysis of frozen pancreatic sections
from IDDM patients revealed that the few remaining insulin-secreting
cells were strongly Fas positive, whereas the
cells from NHP were negative (Fig. 4). The cellular infiltrate in the diabetic pancreata was composed of a minor
population of CD14-positive cells with the morphology of
macrophages, and a number of CD3-positive lymphocytes
(Fig. 4). The majority of islet-infiltrating T cells were CD4
positive, whereas CD8-positive cells were occasionally observed (14). All infiltrating T lymphocytes were perforin
negative (Fig. 4), suggesting that, at the time of our analysis, perforin-mediated T lymphocyte cytotoxicity was not
playing a major role in the destruction of the pancreatic
cells.
Fig. 4.
Fas expression on human pancreatic islet cells from NHP and newly diagnosed IDDM patients. Double immunohistochemical analysis of
pancreas cryostat serial sections (top) exposed to anti-Fas mAb revealed by immunoenzymatic APAAP complex (red), and antiglucagon antibody revealed
by peroxidase-anti-peroxidase complex (brown), shows that Fas is specifically expressed on the non-glucagon-stained, insulin-producing cells. CD3 and
CD14 expression (middle, red) on leukocyte subpopulations (arrows) present in pancreas cryostat sections from IDDM patients. Reactivity of pancreatic islets from an IDDM patient (bottom left) and of the cellular infiltrate within the heart of a patient with myocarditis as a positive control (arrows, bottom right)
with an antiperforin antibody.
[View Larger Version of this Image (121K GIF file)]
Cells Located in Proximity to Reactive FasL+ T Lymphocytes Infiltrating the Diabetic
Pancreata.
cells that
appears to proceed from the islet periphery (as shown in Fig. 5 C) to encompass the entire islet (not shown), most
frequently in proximity to the CD3-positive cells producing FasL.
cells from NHP were consistently Fas negative
and showed no evidence of apoptosis. As expected, apoptosis in infiltrating T lymphocytes was also observed. This
may be the direct consequence of the autoregulated immune process in which T cells, expressing both Fas and
FasL, are not only able to kill Fas positive target cells, but
also themselves or one another (21, 22).
Fig. 5.
Detection of Fas ligand and apoptosis in vivo. (A) FasL expression on human pancreatic sections from NHP and from IDDM patients. (B)
Lane M, marker (100-bp DNA ladder; GIBCO BRL, Gaithersburg, MD). Two primers specific for the FasL coding sequence (5-CCA CCG CCA
CCA CTA CCA-3
, nucleotides 212-229, and 5
-TCT TCC CCT CCA TCA TCA CC-3
, complementary to nucleotides 749-730; These sequence
data are available from EMBL/GenBank/DDBJ under accession number U11821) were selected to specifically amplify FasL cDNA prepared from: lane
1, PMA/ionomycin on activated T lymphocytes from PBLs; lane 2, nonactivated PBLs; lane 3, islets purified from NHP; and lane 4, islets purified from
IDDM patients' pancreata. A 983-bp segment of the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene (Clontech, Palo Alto, CA) was amplified from duplicate aliquots of the same cDNA preparations. (C) In situ TUNEL reaction revealed by 5-bromo-4-chloro-3-indolyl phosphate (black)
staining on IDDM and NHP pancreas sections prelabeled with anti-Fas or anti-insulin mAbs, using 3-amino-9-ethylcarbozole as a substrate (red). Arrows
indicate apoptotic nuclei among Fas- or insulin-positive cells.
[View Larger Version of this Image (80K GIF file)]
cell specific (25). However, the selective expression
of Fas in
cells primed by NO may be responsible for their
specific killing as T cells expressing FasL may promote an
MHC unrestricted destruction of Fas-positive bystander
cells, while sparing neighbor Fas-negative
and
cells. In
accordance with these results, it has been recently demonstrated that transgenic NOD mice expressing FasL in
cells show a higher rate of spontaneous diabetes and are more
sensitive to injection of diabetogenic T cells (9). By contrast, NODlpr/lpr mice, expressing a mutated Fas receptor,
are resistant to both spontaneous and T cell transferred diabetes, suggesting that Fas-induced
cell destruction mediates both human and experimental autoimmune diabetes.
-primed
cells, as high levels of NO
could be released by several periinsulitis cell types, including dendritic cells, macrophages, and endothelial cells (10,
26). The produced NO may directly induce apoptosis in
cells, but NO-induced Fas expression may also mediate a
new mechanism of
cell destruction. The simultaneous
presence in IDDM pancreas of a number of apoptotic Fas-positive
cells and infiltrating FasL-positive lymphocytes suggests a major role for the aberrant Fas expression on
pancreatic
cells in the pathogenesis of human autoimmune diabetes.
Address correspondence to Dr. Massimo Trucco, Children's Hospital of Pittsburgh, Rangos Research Center, 3705 Fifth Ave. at DeSoto St., Pittsburgh, PA 15213, Phone: 412-692-6570, FAX: 412-692-5809; Dr. Carla Giordano, Laboratory of Immunology, Division of Endocrinology, Istituto di Clinica Medica, Universita' di Palermo, Piazza dell Cliniche 2, 90127 Palermo, Italy, Phone: 39-91-655-2110, FAX: 39-91-655-2109; or Dr. Roberto Testi, Department of Experimental Medicine and Biochemical Sciences, University of Rome "Tor Vergata", Via Tor Vergata 135, 00133 Rome, Italy, Phone: 39-6-7259-6502, FAX: 39-6-7259-6505.
Received for publication 19 November 1996 and in revised form 24 July 1997.
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