Cryptosporidium parvum induces
apoptosis in biliary epithelia by a Fas/Fas ligand-dependent
mechanism
Xian-Ming
Chen1,
Gregory J.
Gores1,
Carlos V.
Paya2, and
Nicholas F.
LaRusso1
1 Center for Basic Research in
Digestive Diseases, Division of Gastroenterology and Hepatology,
and 2 Division of Experimental
Pathology, Mayo Medical School, Clinic and Foundation, Rochester,
Minnesota 55905
 |
ABSTRACT |
Although the
clinical features of sclerosing cholangitis from opportunistic
infections of the biliary tree in patients with acquired
immunodeficiency syndrome (AIDS) are well known, the mechanisms by
which associated pathogens, such as Cryptosporidium parvum, cause disease are obscure. Using an in vitro
model of biliary cryptosporidiosis, we observed that
C. parvum induces apoptosis in
cultured human biliary epithelia. Both caspase protease inhibitors and
neutralizing antibodies to either Fas receptor (Fas) and Fas ligand
(FasL) inhibited this process; neutralizing antibodies to other
apoptotic cytokines [interleukin-1
(IL-1
), tumor necrosis
factor-
(TNF-
), and transforming growth factor-
(TGF-
)] had no effect. C. parvum stimulated FasL membrane surface translocation,
increased both FasL and Fas protein expression in infected biliary
epithelia, and induced a marked increase of soluble FasL (but not
IL-1
, TNF-
, and TGF-
) in supernatants from infected cells.
When a coculture model is used in which infected and uninfected cell
populations were physically separated by a semipermeable membrane, both
uninfected biliary epithelia and uninfected Fas-sensitive Jurkat cells
(but not a Fas-resistant Jurkat cell line) underwent apoptosis when
cocultured with infected biliary epithelia. Moreover, both a
neutralizing antibody to FasL and a metalloprotease inhibitor blocked
the apoptosis in uninfected cocultured cells. Activation of caspase
activity was also observed in uninfected cocultured biliary epithelia.
The data suggest that C. parvum
induces apoptosis in biliary epithelia by a Fas/FasL-dependent mechanism involving both autocrine and paracrine pathways. These observations may be relevant to both the pathogenesis and therapy of
the cholangitis seen in AIDS patients with biliary cryptosporidiosis.
acquired immunodeficiency syndrome; cholangiopathies; opportunistic
infections; parasitic diseases; caspase
 |
INTRODUCTION |
ALTHOUGH A COMMON CAUSE of diarrhea in humans and
animals, Cryptosporidium
parvum is usually self-limited in
immunocompetent individuals (37). However, in
immunosuppressed patients, particularly those with the acquired
immunodeficiency syndrome (AIDS), C. parvum may be life threatening (10). Currently, there
is no effective medical treatment for cryptosporidiosis (10). AIDS
patients infected with C. parvum also
develop extraintestinal disease, most frequently of the biliary tract,
resulting in sclerosing cholangitis and cholecystitis in some patients
(10). Indeed, C. parvum may be found
in the bile or the intestine of 20-65% of patients with this
so-called "AIDS-associated cholangiopathy" (2, 5, 11, 56, 59).
The presence of biliary cryptosporidiosis in AIDS patients is
associated with chronicity of infection as well as a poor prognosis
(28).
Although the clinical and radiological features of biliary
cryptosporidiosis have been well documented (59), the
pathophysiological mechanisms underlying C. parvum infection of biliary epithelia are not well
understood. In a previous study, we reported the development of an in
vitro model of biliary cryptosporidiosis in which cultured human
biliary epithelia were infected with C. parvum sporozoites (6). Using this model, we observed
that C. parvum was directly cytopathic
for biliary epithelia, with widespread apoptosis of biliary cells
within hours after exposure (6). The data reported here show that
C. parvum induces apoptosis in
cultured human biliary epithelia by a mechanism involving the Fas
receptor (Fas)/Fas ligand (FasL) system.
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MATERIALS AND METHODS |
C. parvum.
C. parvum oocysts harvested from
calves inoculated with a strain originally obtained from Dr. Harley
Moon at the National Animal Disease Center (Ames, IA) were purchased
from a commercial source (Pleasant Hill Farms, Troy, ID). Oocysts were
purified using a modified ether extraction technique and then suspended in PBS and stored at 4°C. Before infecting biliary epithelial cells, oocysts were treated with 1% sodium hypochlorite on ice for 20 min and subjected to an excystation solution consisting of 0.75%
taurodeoxycholate and 0.25% trypsin for 30 min at 37°C. The
excystation rate was calculated as previously described by others (4,
30) and was determined for each new batch of oocysts.
Cells.
H69 cells (a gift of Dr. D. Jefferson, Tufts University, Boston, MA)
are SV40 transformed human bile duct epithelial cells originally
derived from normal liver harvested for transplant. The cells continued
to express biliary epithelial cell markers, including cytokeratin 19,
-glutamyl transpeptidase, and ion transporters, consistent with
biliary function and have been extensively characterized (15). Stock
cultures of these nonmalignant but immortalized cells were maintained
in coculture with irradiated NIH3T3 mouse fibroblasts and were grown in
a hormonally supplemented medium with 10% fetal bovine serum. For
experiments, cells were maintained for three passages without coculture
cells to ensure that the culture was free of fibroblasts. All
experiments were performed with cells between passage
23 and 26. Jurkat
E6-1, a human leukemia T cell that naturally expresses Fas, was
obtained from the American Type Culture Collection (ATCC, Rockville,
MD). Jurkat JM-3A5, a human leukemia T cell that does not express Fas
(a gift from Dr. Paul Leibson, Rochester, MN) was used as negative
control for Fas/FasL-based cytotoxicity induced by C. parvum-infected H69 monolayers. Jurkat cell lines were
cultured in RPMI 1640 medium containing 10% FCS, 2 mM
L-glutamine, 100 U/ml
penicillin, and 100 µg/ml streptomycin.
Infection models.
Infections were performed as described previously (6). Briefly, H69
cells were seeded into four-well chamber slides or six-well Costar
tissue culture plates (Becton Dickson Labware) and grown to
70-80% confluence. Infection with C. parvum was accomplished in a culture medium consisting
of DMEM-F12, 100 U/ml penicillin, and 100 µg/ml streptomycin
(referred to hereafter as assay medium) and freshly excysted
C. parvum sporozoites. Inactive
organisms (excysted sporozoites treated at 65°C for 2 h) were used
for sham infection controls. The viability of each oocyte preparation
was determined prior to each set of experiments, and an optimal dose of
oocysts and sporozoites was determined. In most experiments, a
concentration of 1-5 × 106 sporozoites was used.
Apoptotic cytotoxicity of C. parvum to
H69 cells.
Subconfluent (70-80% confluency) H69 cells in four-well chamber
slides were used. Before C. parvum
infection, cells were washed with DMEM-F12 and then incubated in 0.3-ml
assay medium containing freshly excysted C. parvum sporozoites. Cells were either incubated with 1 × 106 C. parvum sporozoites per well for different periods of
time or incubated with C. parvum at
concentrations from 1 × 105
to 5 × 106 sporozoites per
well for 24 h. After incubation, slides were fixed in absolute methanol
for 5 min, and the degree of apoptosis was evaluated by
4,6-diamidino-2-phenylindole (DAPI) staining and fluorescein-labeled
annexin V (Pharmingen, San Diego, CA) binding.
Inhibition of apoptotic cytotoxicity by antibodies.
To assess for potential inhibition of apoptotic cytotoxicity of
candidate apoptotic factors, neutralizing antibodies against interleukin-1
(IL-1
; 50 µg/ml; R&D Systems, Minneapolis, MN), transforming growth factor-
(TGF-
; 50 µg/ml; R&D Systems),
tumor necrosis factor-
(TNF-
; 50 µg/ml, R&D Systems) and FasL,
NOK-1 (17, 23) (10 µg/ml, Pharmingen), as well as Fas antagonistic antibody M3 (31, 33, 34) (10 µg/ml, Immunex, Seattle, WA), were added
to H69 cells grown on four-well chamber slides. M33 (Immunex), an
isotype-matched irrelevant monoclonal antibody to M3, and normal goat
and chicken IgG were used as control antibodies. After removal of the
culture media, H69 cells were washed with DMEM-F12 and resuspended in
0.3-ml assay medium containing those antibodies. Thirty minutes after
the addition of antibodies, freshly excysted C. parvum sporozoites were added. After incubation for 24 and 48 h, cells were fixed and apoptosis was assayed. In some experiments, cells were incubated with antibodies and
C. parvum sporozoites for 2 h and then
washed three times with PBS and fixed in absolute methanol for 5 min.
Parasites that had attached to or that had invaded H69 cells were
quantitated by immunofluorescence using a monoclonal antibody against a
sporozoite protein (2H2; ImmuCell, Portland, ME) (46).
Inhibition of apoptotic cytotoxicity by YVAD-CHO and DEVD-CHO.
H69 cells were grown on four-well chamber slides to 70-80%
confluence. Prior to infection of C. parvum sporozoites, the medium was replaced with the
assay medium, and the cells were incubated for 30 min with the cell
permeable caspase inhibitors DEVD-CHO (2 µM; Biomol, Plymouth
Meeting, PA) and YVAD-CHO (2 µM; Biomol). Infection of
C. parvum sporozoites was performed as
previously described. After 24 and 48 h of incubation, cells were fixed
and apoptosis was determined. In some experiments, cells were only incubated with C. parvum sporozoites
for 2 h and then fixed with methanol, and infection rates were
determined by immunofluorescence as previously described.
Expression of FasL and Fas in infected H69 monolayers.
H69 cells were grown to 70-80% confluency in T25 flasks and then
exposed to C. parvum in the assay
medium. After different incubation times, cells were lysed and
quantitative immunoblots were performed as previously described (26,
29, 43). Briefly, samples were analyzed by SDS-PAGE, and separated
proteins were transferred to nitrocellulose membranes. Membranes were
sequentially incubated with primary antibodies and then with 0.2 µg/ml of horseradish peroxidase-conjugated secondary antibody and
revealed by an enhanced chemiluminescence detection system (Amersham,
Buckinghamshire, England). G247-4 (Pharmingen) antibody against
FasL, which recognizes both the membrane bound and soluble forms of
FasL (38, 39), and clone 13 (Transduction Laboratories, Lexington, KY)
against Fas were used for the immunoblotting. For immunocytochemistry of surface membrane expression of Fas and FasL, H69 cells were grown to
70-80% confluency on four-well chamber slides and then exposed to
C. parvum. In some slides, 0.2 mM
1,10-phenanthroline (Sigma, St. Louis, MO), a metalloprotease inhibitor
(23) that at a dose of 0.2 mM showed no toxicity to H69 cells, was
added to the assay medium at the same time as sporozoites. After 24 h
of incubation, cells were fixed with 0.1 M PIPES (pH 6.95), 1 mM
[ethylene-bis(oxyethylenenitrilo)]tetraacetic acid, 3 mM MgSO4 and 2% paraformaldehyde.
Without membrane permeabilization, cells were incubated with primary
monoclonal antibodies against Fas (clone 13) or FasL (G247-4)
followed by fluorescein-labeled anti-mouse antibodies. Slides were
mounted with mounting medium (H-1000, Vector Laboratories) and assessed
by confocal laser scanning microscopy. Contrast and intensity for each
image were manipulated uniformly using Adobe (Mountain View, CA)
Photoshop software.
Detection of soluble FasL, IL-1
,
TGF-
, and TNF-
.
To analyze for the production of potential soluble apoptotic factors
released by H69 cells in response to C. parvum infection, H69 cells were grown to subconfluence
in T75 flasks. After different periods of incubation with assay medium
containing freshly excysted sporozoites, supernatants were collected
for assays. To detect soluble FasL (sFasL), 15 ml of supernatants were
concentrated to 0.15 ml using an ultrafree concentrator with 10,000 molecular weight limits after 1 h of spinning at 3,000 rpm at 4°C.
The concentrated supernatants were then mixed 1:1 with lysis buffer
[50 mM Tris, containing 5 mM EDTA, 0.2 mM phenylmethylsulfonyl
fluoride (PMSF), 1 µg/ml pepstatin, and 0.5 µg/ml leupeptin
adjusted to pH 7.4]. The concentrated supernatants were analyzed
by SDS-PAGE and immunoblotting as previously described. G247-4
antibody, which recognizes both the membrane bound and soluble forms of
FasL (38, 39), was used. IL-1
, TNF-
, and TGF-
concentrations
in the supernatants were determined by ELISA using commercial kits (R&D Systems).
Apoptotic cytotoxicity of recombinant human sFasL to H69 cells.
H69 cells were grown to 70-80% confluency on four-well chamber
slides and then exposed to recombinant human sFasL (Calbiochem, Cambridge, MA) at concentrations from 100 to 1,000 ng/ml in the assay
medium. After 10 h of incubation, cells were fixed and apoptosis was analyzed.
Apoptotic cytotoxicity of C. parvum-infected H69 monolayers for uninfected cocultured
cells.
Cytotoxicity of C. parvum- infected
H69 monolayers for noninfected cells was evaluated by using a coculture
system. For the H69/H69 coculture system, H69 cells were grown to
70-80% confluency in six-well Costar tissue culture inserts
(Becton Dickinson Labware) with cells both on the inserts (upper
chamber) and on the plates below the inserts (lower chamber). The two
cell populations were physically separated by a polycarbonate membrane
with a high density of 0.4-µm pore size, which allows free exchanges
of molecules (but not sporozoites) between the upper and lower media
reservoirs. After removal of H69 culture medium, cells were washed with
DMEM-F12 and resuspended in assay medium. Sporozoites were then added
to the assay medium of the upper chamber. In some experiments, 0.2 mM
1,10-phenanthroline was added to the medium in the upper chamber at the
same time as sporozoites. After 24 h of incubation, cells in the upper
and lower chambers were fixed and apoptosis was evaluated. In control
experiments, sporozoites were added to the upper chamber of the inserts
without H69 cells and then cocultured with H69 cells grown on the
plates in the lower chamber.
For the coculture of H69 with Jurkat cell lines, the same coculture
system was applied, but the upper chambers were seeded with uninfected
Jurkat cells instead of H69 cells. After removal of cell culture
medium, both cell populations were resuspended in the assay medium and
freshly excysted C. parvum sporozoites were added to the H69 cells grown in the lower chamber. In some experiments, the FasL neutralizing antibody NOK-1 (10 µg/ml,
Pharmingen) was added in the lower chamber 30 min before the addition
of C. parvum sporozoites. After 24 h
of incubation, H69 cells were fixed and apoptosis was determined by
DAPI staining. Jurkat cells were resuspended in the media and applied
to slides using a Cytospin (Shandon, 1,000 rpm, 6 min). Cells on the
slides were then fixed and stained with DAPI.
Caspase activity in cocultured uninfected H69 cells.
H69 cells were grown to 70-80% confluency in six-well Costar
tissue culture inserts with cells both on the inserts (upper chamber)
and on the plates below the inserts (lower chamber) as previously
described. Sporozoites were then added to the assay medium of the upper
chamber. After different periods of incubation, cocultured H69 cells on
the plates were washed with PBS, and the cytosolic extracts were
prepared by using a lysis buffer [25 mM HEPES (pH 7.5), 5 mM
magnesium chloride, 1 mM EGTA, 0.5 mM PMSF, 2 µg/ml leupetin, and 2 µg/ml pepstatin]. Cell lysate was incubated at 37°C for 30 min in assay buffer (100 mM HEPES, pH 7.5, 10% sucrose, 10 mM
dithiothreitol, and 0.5 mM EDTA) with 20 µM
Ac-DEVD-aminotrifluoromethyl coumarin (AFC) as a caspase fluorescent
substrate (Enzyme Systems Products, Livermore, CA). Fluorescence at
390-475 nm was measured with a Perkin-Elmer fluorescence
spectrophotometer (Buckinghamshire, UK). Measurements were calibrated
against a standard curve of AFC (Enzyme Systems Products), and data
were expressed in picomoles of released AFC per milligram of lysate
proteins. Protein concentration was measured by Bradford method
(Bradford reagent, Sigma).
Apoptosis measurement.
Apoptosis was quantitated by DAPI staining and annexin V binding. Cells
were stained with the nuclear staining dye DAPI (2.5 µM, 5 min) or
fluorescein-labeled annexin V (Pharmingen) and viewed with a
fluorescence microscope. For each well on the slide, over 2,000 cells
were counted, and the number of cells positive to annexin V binding or
DAPI staining with nuclear changes characteristic of apoptosis (i.e.,
condensation, margination, and/or fragmentation) was recorded (9, 21,
42). Apoptosis was also assessed by DNA extraction and agarose
electrophoresis. DNA was extracted from the cultured cells using a
phenol-chloroform technique, with ethidium bromide added and run on an
agarose gel. Bands were visualized and photographed under ultraviolet
light (42).
Statistical analysis.
All values are given as means ± SE. Means of groups were compared
with Student's (unpaired) t-test or
ANOVA test where appropriate. P < 0.05 was considered statistically significant.
 |
RESULTS |
Apoptosis of C. parvum-infected human
cholangiocytes.
When stained with the nuclear binding dye DAPI, H69 cells in infected
monolayers grown either on chamber slides or insert membranes exhibited
the characteristic nuclear changes associated with apoptosis of
epithelial cells to a significantly greater extent than did cells in
the sham infection and normal controls (Fig.
1). Evidence of apoptosis in infected H69
cells was further confirmed by annexin V binding, which showed a
similar dose-dependent increase of apoptotic cells after incubation
with various concentrations of C. parvum for 24 h (Fig.
1A); an identical staining pattern of apoptotic cells with DAPI staining (Fig.
1C) was found. The number of
apoptotic cells in infected H69 cell monolayers grown on four-well
chamber slides increased steadily after 24 h of incubation when up to
1 × 106
C. parvum sporozoites per well were
added and increased consistently up to 22% when incubated with 5 × 106 C. parvum sporozoites per well. When cells were incubated
with 1 × 106
C. parvum sporozoites per well,
cytopathic effects became apparent by 12 h after exposure to the
organism and increased to 12% apoptotic cells at 24 h, compared with
<1% apoptosis in uninfected or sham-infected cells over the same
period (Fig. 1B). Therefore, in most
of the subsequent experiments, a concentration of 1 × 106 C. parvum sporozoites per four-chamber slide well was
used.

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Fig. 1.
Apoptosis in H69 cells induced by Cryptospordium
parvum infection. A:
infection causes dose-dependent apoptosis in infected H69 monolayers.
Both 4,6-diamidino-2-phenylindole (DAPI) staining and
fluorescein-labeled annexin V binding showed a similar dose-dependent
increase of apoptotic cells after incubation with C. parvum for 24 h. B:
infection causes a time-dependent apoptosis in infected H69 monolayers
by DAPI staining. * P < 0.01 compared with sham
infection. C: apoptosis in
C. parvum-infected H69 monolayers
observed by DAPI and annexin V staining. Nuclei of infected cells at 24 h postinfection show fragmentation of DNA and segmentation of nucleus
(arrowheads) with fluorescent DNA binding dye DAPI, which is consistent
with annexin V binding. No significant number of cells showing
segmentation of nucleus or fragmentation of DNA for DAPI staining or
positive to annexin V binding was found in cells of sham-infected
controls. Bars = 5 µm.
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Inhibition of apoptosis by antibodies and caspase inhibitors.
To explore possible mechanisms of C. parvum-induced apoptosis in infected H69 cells,
parallel experiments were performed in the presence of various
antibodies that could block Fas/FasL interactions or neutralize FasL,
IL-1
, TNF-
, and TGF-
activities. As shown in Fig.
2A,
exposure of H69 cells to a Fas antagonistic antibody (M3) or a FasL
neutralizing antibody (NOK-1) significantly
(P < 0.01) reduced apoptosis induced
by C. parvum infection; in contrast, antibodies neutralizing IL-1
, TNF-
, and TGF-
showed no effect on C. parvum-induced apoptosis. All
antibodies used in the experiments had no effect on the rates of
infection of H69 cells by C. parvum sporozoites (data not shown). Pretreatment of cells with the caspase inhibitors DEVD-CHO and YVAD-CHO also blocked C. parvum-induced apoptosis up to 80% (Fig.
2B). These observations suggested
that Fas/FasL interactions are the major factors involved in the
mechanisms of C. parvum-induced
apoptosis.

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Fig. 2.
Inhibition of apoptosis in C. parvum-infected H69 monolayers. Thirty minutes before
addition of C. parvum sporozoites,
antibodies or caspase protease inhibitors were added to the culture
media. Apoptosis in infected H69 cells after 24 and 48 h incubation
with C. parvum was assayed by DAPI
staining. A: Fas receptor (Fas) and
Fas ligand (FasL) neutralizing antibodies, but not antibodies against
interleukin-1 (IL-1 ), tumor necrosis factor- (TNF- ), and
transforming growth factor- (TGF- ), significantly reduced the
apoptosis induced by C. parvum.
B: caspase inhibitors DEVD-CHO and
YVAD-CHO also inhibited apoptosis induced by C. parvum. * P < 0.01 compared with controls. Ab, antibody.
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Upregulation of Fas/FasL in human cholangiocytes by C. parvum.
The presence of performed FasL and Fas in H69 cells was assessed by
quantitative Western blotting using monoclonal antibodies against human
FasL and Fas. Cell lysates from H69 cells showed a band of 37-kDa
molecular mass (Fig.
3A),
consistent with other reports of FasL (23, 38, 39). The intensity of
FasL band decreased at 6 h postinfection but increased significantly
(P < 0.001) at 12 and 24 h
postinfection (Fig. 3, A and
B). Cell lysates from noninfected
H69 cells contained detectable amounts of Fas, with a molecular mass of
47 kDa, and the intensity of this band also increased significantly at
24 h postinfection (Fig. 3, C and
D). The membrane
surface expression of Fas and FasL in infected H69 monolayers were
assessed by immunocytochemistry and laser confocal microscopy without
cell membrane permeabilization. H69 cells showed a distinct surface
staining for Fas in sham-infected controls, but no staining for FasL
was observed (Fig. 4). After incubation
with C. parvum for 24 h, intensive
surface staining for both Fas and FasL was detected. FasL surface
expression on infected cells was further augmented by the presence of
metalloprotease inhibitor 1,10-phenanthroline (Fig. 4). These
observations suggest that both FasL and Fas are normally expressed in
H69 cells, but whereas Fas is on the surface of uninfected cells, FasL
is not normally found on the cell surface of cholangiocytes. In
contrast, C. parvum infection not only
increases Fas and FasL expression but also induces the translocation of
FasL to the membrane and stimulates the cleavage of membrane FasL to
form sFasL.

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Fig. 3.
Expression of Fas and FasL in C. parvum-infected H69 monolayers by immunoblotting. After
incubation with C. parvum sporozoites,
H69 cells were lysed and subjected to quantitative immunoblotting.
A and
C: representative immunoblots for FasL
and Fas in H69 cells. B and
D: densitometric analysis of Fas and
FasL expression from 3 separate experiments. -Actin was also
immunoblotted to ensure equal loading of proteins to each lane.
* P < 0.01 compared with sham
infection.
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Fig. 4.
Membrane surface expression of Fas/FasL on C. parvum-infected H69 monolayers by immunocytochemistry
and laser confocal microscopy. H69 cells showed distinct surface
staining for Fas in sham-infected controls, but intensive staining for
Fas was found in cells at 24 h postinfection. No significant positive
reaction to FasL was found in sham-infected control cells in presence
or absence of metalloprotease inhibitor 1,10-phenanthroline. In
contrast, positive staining for FasL was observed in cells 24 h
postinfection, and positive reaction to FasL was further augmented by
presence of metalloprotease inhibitor. Bars = 5 µm.
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Release of sFasL from C. parvum-infected
human cholangiocytes.
To determine if sFasL was present in the supernatants of
C. parvum-infected H69 monolayers,
supernatants from C. parvum-infected H69 cells were collected and analyzed by quantitative Western blotting.
The immunogen of G247-4 is the COOH terminal of the FasL molecule,
and this antibody recognizes both the membrane bound (FasL) and sFasL
forms (38, 39). Although not detectable in the supernatants from
sham-infection controls, sFasL increased steadily in the 24-h
supernatants from infected H69 monolayers (Fig.
5, A and
B). No significant increase of
IL-1
, TNF-
, and TGF-
in the supernatants of
C. parvum-infected H69 monolayers was
found compared with noninfected or sham-infected controls using ELISA
assays (Fig. 5C).

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Fig. 5.
Soluble FasL (sFasL), IL-1 , TGF- , and TNF- in supernatants of
C. parvum-infected H69 monolayers.
After 24 h of incubation with C. parvum sporozoites, supernatants were collected for
cytokine determination. A:
representative immunoblot for sFasL in supernatants after concentration
with ultrafree concentrator. Supernatants from C. parvum-infected H69 monolayers showed strong band for
sFasL, whereas no positive band was found in the supernatants from sham
controls. B: densitometric analysis of
sFasL in supernatants. C: IL-1 ,
TGF- , and TNF- levels in supernatants determined by ELISA.
No significant difference in their values of the supernatants was found
between C. parvum infection and sham infection. Data were
from 3 separate experiments. * P < 0.01 compared
with sham infection.
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Apoptotic cytotoxicity of C. parvum-infected human cholangiocytes to
cocultured H69 cells.
To test the potential importance of sFasL in regulation of apoptosis in
C. parvum-infected H69 cultures,
cytotoxicity of C. parvum-infected H69
monolayers was further evaluated using a coculture system. H69 cells
grown in the lower chamber in the coculture system, which were not
directly infected with C. parvum, also showed characteristic changes of apoptosis (Fig.
6A).
Apoptosis of uninfected H69 cells cocultured with infected H69 cells
was further confirmed by DNA extraction and agarose gel
electrophoresis, which produced a ladder-like pattern from the DNA of
those cells (Fig. 6B). H69 cells
cocultured with C. parvum sporozoites
alone in the upper chamber (no H69 cells grown on the insert) showed no
cells undergoing apoptosis (data not shown).

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Fig. 6.
Apoptosis in cocultured H69 cells and cytotoxicity of recombinant human
sFasL to H69 cells. A: both infected
(upper chamber) and uninfected (lower chamber) H69 showed apoptosis
after 24 h of coculture. Addition of 0.2 mM 1,10-phenanthroline to
upper chamber at same time as C. parvum was added completely blocked apoptosis in
cocultured uninfected H69 cells. In contrast, a significant increase of
apoptosis in infected cells in presence of inhibitor was found.
B: ladder pattern indicative of
internucleosomal cleavage characteristic of apoptosis was seen when DNA
was extracted from C. parvum-infected
monolayer (lane 2) or cocultured
uninfected cells (lane 3) and
subjected to agarose gel electrophoresis; lane
1 contains molecular weight markers (100 bp DNA
ladder). This laddering was not seen in the sham-infection control
cells when equal amounts of DNA was loaded (sham-infected cells,
lane 4; cocultured cells,
lane 5).
C: recombinant human sFasL induces
apoptosis in H69 cells. Incubation of cells with recombinant human
sFasL for 10 h at a dose of 100-1,000 ng/ml resulted in
significant apoptosis in H69 cells in a dose-dependent manner.
* P < 0.01 compared with
normal control. ** P < 0.05 compared with infection.
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The role of sFasL in C. parvum-induced
apoptosis in H69 cells was further confirmed by using a metalloprotease
inhibitor in the H69/H69 coculture system. The metalloprotease
inhibitor, 1,10-phenanthroline, which prevents the cleavage of sFasL
from cell membranes, completely blocked the apoptosis in cocultured
uninfected H69 cells (Fig. 6A). In
contrast, an increase of apoptosis in H69 cells directly infected with
C. parvum was found in the presence of
this inhibitor. Furthermore, incubation of H69 cells with recombinant
human sFasL for 10 h resulted in significant apoptosis in cells in a
dose-dependent manner (Fig. 6C).
Activation of caspase family in cocultured biliary epithelia.
To further confirm the role of Fas/FasL pathway in the mechanisms of
apoptosis induced by C. parvum,
cytosolic extracts were prepared from H69 cells cocultured with
C. parvum-infected biliary epithelia,
and caspase activity in the lysates was determined using a fluorescent
substrate Ac-DEVD-AFC. A time-dependent increase of caspase activity
was detected in those cocultured-uninfected H69 cells (Fig.
7).

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Fig. 7.
Activation of caspase activity in H69 cells cocultured with
C. parvum-infected H69 cells. After
different periods of coculture, uninfected H69 cells on the plates were
harvested, and lysates were prepared and assayed. Caspase activity was
estimated with caspase substrate Ac-DEVD-AFC. Activation of caspase
activity was found in cells cocultured with C. parvum-infected H69 cells. Each point represents mean
values of triplicates of 2 separate experiments.
* P < 0.01 compared
with controls at time 0.
|
|
Apoptotic cytotoxicity of C. parvum-infected human cholangiocytes to
cocultured Fas-sensitive Jurkat cells.
To further confirm that Fas/FasL interactions are involved in
C. parvum-induced apoptosis, we
designed cytotoxicity tests using our coculture system in which
C. parvum-infected H69 monolayers were
used as effectors and noninfected Jurkat cells as targets, in the
presence or absence of the FasL neutralizing antibody NOK-1. As shown
in Fig. 8, about 14% of FasL sensitive
Jurkat E6-1 cells underwent apoptosis after 24 h of coculture with
C. parvum-infected H69 monolayers, and
the FasL neutralizing antibody NOK-1 completely blocked the apoptosis
(Fig. 8). In contrast, no increase of apoptosis above control was found
for the Fas-resistant Jurkat JM-3A5 cells cocultured with
C. parvum-infected H69 monolayers
(Fig. 8). These observations suggested that sFasL was being released
from C. parvum-infected H69 cells and
was inducing apoptosis of Fas-sensitive cocultured cells.

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|
Fig. 8.
Apoptosis in Jurkat cells cocultured with C. parvum-infected H69 monolayers. Jurkat cells were
cocultured with C. parvum-infected H69
cells. After 24 h of coculture, Jurkat cells were fixed and
apoptosis was assayed by DAPI staining. Fas-sensitive Jurkat cells
(Jurkat E6-1), but not Fas-resistant Jurkat cells (Jurkat JM-3A5),
cocultured with C. parvum-infected H69
cells showed characteristic changes of apoptosis. FasL neutralizing
antibody completely blocked the apoptosis.
* P < 0.01 compared with
normal control.
|
|
 |
DISCUSSION |
The results of our studies provide the first evidence that the Fas/FasL
system is involved in the cytotoxicity of C. parvum for any epithelia. Our data show
that 1) C. parvum infection of cultured human biliary epithelia
results in apoptosis of the infected monolayers, a process that is
blocked both by caspase protease inhibitors and by Fas or FasL
neutralizing antibodies; 2)
C. parvum infection induces membrane
surface translocation of FasL and stimulates Fas and FasL protein
expression in infected human biliary epithelial cells;
3) C. parvum infection releases sFasL from infected
monolayers; and 4) uninfected
biliary epithelia or Fas-sensitive Jurkat cells undergo apoptosis via a
Fas/FasL dependent pathway when cocultured with infected biliary
epithelia. The data suggest that C. parvum is cytotoxic for biliary epithelial cells via a
mechanism involving Fas/FasL activation and provide insight into the
potential pathogenic mechanisms whereby C. parvum causes biliary tract disease.
Apoptosis plays a critical role in the regulation of inflammation and
in the host immune response. Recent data in other tissues infected with
either parasites (such as Entamoeba
histohytica, Schistosoma
mansoni, Trypanosoma
cruzi, and Toxoplasma
gondii) or bacteria are consistent with the concept
that microbial pathogens can kill cells by an apoptotic mechanism (8,
24, 27, 45, 57, 58, 61). However, the cellular mechanisms by which
individual pathogens induce apoptosis in specific host cells,
especially epithelial cells, remain obscure. For some pathogens (e.g.,
Shigella, Salmonella, E. histolytica) (45, 61), the cells undergoing apoptosis
are limited to those directly infected by the pathogen. For other
pathogens, e.g., human immunodeficiency virus (HIV) and herpes virus 6 (18, 22), the cells undergoing apoptosis are distinct from those that
are infected; in this later instance, the Fas/FasL system has been
mechanistically implicated (1, 7, 18, 22). Based on our data, it
appears that C. parvum, like HIV and
herpes virus 6, can initiate apoptosis in cells like biliary epithelia
remote from those cells actually infected by the organism employing
Fas/FasL.
The Fas (APO-1 and CD95)/Fas-L system has emerged as an important
cellular pathway regulating the induction of apoptosis in a variety of
tissues (1, 11, 14, 60). Fas is a widely expressed, 45-kDa type I
membrane protein of the TNF-nerve growth factor family of cell surface
receptors. In cells expressing Fas, apoptosis occurs after Fas
interaction with 1) its natural
ligand FasL, a 37-kDa type II protein;
2) agonistic anti-Fas antibody; or
3) sFasL, a biologically active form
of FasL released from cell membranes by a metalloprotease (20, 23, 35,
50, 54). By activating a variety of downstream effector cellular
proteases, including members of the caspase family, Fas induces
apoptosis (35, 36). The biological importance of the Fas/FasL system has been extensively studied in T cells, where it plays a critical role
in the clonal deletion of autoreactive T cells and in the activation-induced suicide of T cells (20, 35, 36). Fas/FasL is also
extensively expressed in epithelial cells and mediates apoptosis in
epithelia in a variety of organs (3, 13, 32, 41, 44, 48). In the
digestive tract, the Fas/FasL pathway has been reported to play a role
in the apoptosis of colonic epithelial cells in ulcerative colitis
(52), in gastric epithelial cells infected by
Helicobacter pylori (47), and in
hepatocytes in acute Wilson's disease (51). Biliary epithelial cells
have been shown to express Fas by immunohistochemistry in primary
biliary cirrhosis, primary sclerosing cholangitis (25), and in
noncancerous lesions of hepatocellular carcinoma patients with chronic
hepatitis or liver cirrhosis (16, 19).
To test the potential importance of the Fas/FasL pathway in regulation
of apoptosis in C. parvum-infected
biliary epithelia, we examined the effects of antagonistic anti-Fas,
neutralizing anti-FasL antibodies, and caspase inhibitors on
C. parvum-induced apoptosis in
cultured biliary epithelia. Both types of antibodies as well as caspase
inhibitors significantly reduced apoptosis in C. parvum-infected H69 monolayers by up to 80%. Our data
also showed that C. parvum infection
can stimulate cytoplasmic FasL membrane surface translocation and
induce the expression of Fas and FasL in infected biliary monolayers.
Upregulation of Fas and FasL proteins, coupled with the
C. parvum-induced FasL membrane translocation and release of sFasL (an event blocked by a
metalloprotease inhibitor) should adequately explain the accelerated
apoptosis of both the biliary epithelia directly infected with
C. parvum and those cocultured with
the infected cells.
Thus it appears that FasL may function in either autocrine or paracrine
pathways to produce apoptosis in this model (Fig. 9). FasL translocated to the membrane from
the cytoplasm can interact with Fas on adjacent cells, activate the
pathway and therefore induce apoptosis. Indeed, whereas the
metalloprotease inhibitor completely blocked the apoptosis in
cocultured uninfected biliary epithelia, an increase of apoptosis in
cells directly infected with C. parvum
was found, suggesting FasL accumulation on the cell membrane surface in
the presence of an inhibitor that prevents the cleavage of FasL to form
sFasL. Using the coculture system, we found that C. parvum-infected biliary epithelia could induce either
other biliary epithelia or Fas-sensitive Jurkat T cells to undergo
apoptosis and that the apoptosis was blocked by both FasL neutralizing
antibody and by a metalloprotease inhibitor. sFasL in the supernatant
of C. parvum-infected biliary
epithelial cells increased, whereas other apoptotic cytokines, such as
IL-1
, TNF-
, and TGF-
, showed no significant change. Activation
of caspase activity in uninfected biliary epithelial cells was also found when cocultured with C. parvum-infected biliary epithelia. These results
further support the notion that C. parvum infection of human biliary epithelia induces
uninfected cells to undergo apoptosis via a Fas/FasL dependent
paracrine mechanism.

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|
Fig. 9.
Schematic model of C. parvum-induced
apoptosis in biliary epithelial cells. C. parvum induces Fas and FasL expression in infected
biliary epithelial cells. FasL can be expressed as a membrane-bound
form and mediate apoptosis in a manner of fratricide, interacting with
Fas on neighboring cells. FasL-mediated apoptosis also occurs in an
autocrine (interacting with Fas overexpressed on same cell) or
paracrine fashion (interacting with Fas on bystander cells) via
sFasL.
|
|
Reports about the cytotoxicity of sFasL are conflicting. Our present
observations do not agree with those of Schneider et al. (49). In that
study, investigators found that sFasL released by metalloproteases had
very low Fas-mediated cytotoxicity (1,000 times less than membrane
FasL). Instead, the Fas-mediated cytotoxicity found in the media was
attributed to a large (>500 kDa) product that probably represented
fragments of membrane-bound FasL or vesicles containing membrane-bound
FasL. However, our results are in agreement with other reports that
observed cytotoxicity of sFasL. For example, sFasL released from
stimulated T cells produces apoptosis of Fas-sensitive Jurkat T cells;
also, recombinant sFasL has been reported to induce apoptosis both in
vitro and in vivo (26, 29, 54, 55). Although immortalization of cells
with SV40 may decrease p53 expression and thus increase cellular
resistance to apoptosis, and although sFasL is not apoptotic in all
cell lines in vitro (40, 53), our data show that H69 cells are
relatively sensitive to sFasL-induced apoptosis.
Although uninfected cultured human biliary epithelial cells express
both Fas and FasL, our results with the cocultured biliary epithelial
cells and Fas-sensitive Jurkat T cell line indicate that biliary
epithelial cells mediate apoptosis of target cells only when they are
infected with C. parvum. One possible
explanation for this observation is that FasL may not be localized at
the membrane in noninfected cells and thus is not self-toxic, as
confirmed by our immunocytochemical staining showing the absence of
FasL on the cell membrane surface under unstimulated conditions. This also appears to be the situation in Jurkat cells, which also express both Fas and FasL under unstimulated conditions (29). Soluble factors
elaborated directly from C. parvum are
not likely to play a role in the apoptotic cytotoxicity because biliary
epithelial cells cocultured with C. parvum alone (separated by the insert membrane) do not
undergo accelerated apoptosis.
In summary, using an in vitro model of biliary cryptosporidiosis, we
found that C. parvum induced apoptosis
in infected human biliary epithelial cultures, and C. parvum infection of biliary epithelial cells can
further induce uninfected cells to undergo apoptosis via a
Fas/FasL-dependent mechanism likely involving both autocrine and
paracrine pathways (Fig. 9). Future studies should address the
mechanism(s) by which C. parvum
infection results in cleavage of sFasL from cell membranes and how this
organism increases the expression of Fas and FasL.
 |
ACKNOWLEDGEMENTS |
We thank Drs. F. Que and A. Celli for helpful advice and Dawn
Lubinski and Deb Hintz for excellent secretarial assistance.
 |
FOOTNOTES |
This work was supported by the National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-24031 (N. F. LaRusso) and a
grant from the Mayo Foundation.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: N. F. LaRusso, Center for Basic Research in Digestive Diseases, Mayo Clinic,
200 First St., S.W., Rochester, MN 55905 (E-mail:
larusso.nicholas{at}mayo.edu).
Received 14 January 1999; accepted in final form 9 June 1999.
 |
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