0021-972X/00/$03.00/0
The Journal of Clinical Endocrinology & Metabolism
Copyright © 2000 by The Endocrine Society
Vol. 85, No. 1
Printed in U.S.A.
Mechanisms of Coxsackievirus-Induced Damage to
Human Pancreatic b-Cells*
MERJA ROIVAINEN, SUVI RASILAINEN, PETRI YLIPAASTO, RIIKKA NISSINEN,
JARKKO USTINOV, LUC BOUWENS, DÉCIO L. EIZIRIK, TAPANI HOVI, AND
TIMO OTONKOSKI†
Enterovirus Laboratory, National Public Health Institute (M.R., P.Y., R.N., T.H.), FIN-00300 Helsinki,
Transplantation Laboratory, Haartman Institute (S.R., J.U., T.O.), and Hospital for Children and
Adolescents (T.O.), University of Helsinki, FIN-00014 Helsinki, Finland; and the Diabetes Research
Center, Vrije Universiteit Brussel (L.B., D.L.E.), B-1090 Brussels, Belgium
ABSTRACT
Enteroviruses may be involved in the pathogenesis of insulindependent diabetes mellitus, either through direct b-cell infection or
as triggers of autoimmunity. In the present study we investigated the
patterns of infection in adult human islet cell preparations (consisting
of 56 6 14% b-cells) by several coxsackieviruses. The cells were infected with prototype strains of coxsackievirus B (CBV) 3, 4, and 5 as
well as coxsackievirus A9 (CAV-9). The previously characterized diabetogenic strain of coxsackievirus B4 (CBV-4-E2) was used as a
reference. All viruses replicated well in b-cells, but only CBVs caused
cell death. One week after infection, the insulin response of the b-cells
to glucose or glucose plus theophyline was most severely impaired by
CBV-3 and CBV-5 infections. CBV-4 also caused significant functional impairment, whereas CAV-9-infected cells responded like uninfected controls. After 2 days of infection, about 40% of CBV-5infected cells had undergone morphological changes characteristic of
pyknosis, i.e. highly distorted nuclei with condensed but intact chromatin. Both mitochondria and plasma membrane were intact in these
cells. DNA fragmentation was found in 5.9 6 1.1% of CBV-5-infected
b-cell nuclei (2.1 6 0.3% in controls; P , 0.01). CAV-9 infection did
not induce DNA fragmentation. One week after infection the majority
of infected cells showed characteristics of secondary necrosis. Medium
nitrite and inducible nitric oxide synthase messenger ribonucleic acid
levels were not significantly up-regulated by CBV infection. These
results suggest that several enteroviruses may infect human b-cells.
The infection may result in functional impairment or death of the
b-cell or may have no apparent immediate adverse effects, as shown
here for CAV-9. Coxsackie B viruses cause functional impairment and
b-cell death characterized by nuclear pyknosis. Apoptosis appears to
play a minor role during a productive CBV infection in b-cells. (J Clin
Endocrinol Metab 85: 432– 440, 2000)
I
NSULIN-DEPENDENT diabetes mellitus (IDDM) is
caused by progressive destruction of pancreatic b-cells,
resulting in insulin deficiency. Several lines of epidemiological evidence suggest that enterovirus infections, especially
those due to the group B of coxsackieviruses may have a role
in the etiology of IDDM (1–9). According to some studies,
outbreaks of IDDM may be associated with enterovirus epidemics (10, 11). Results from prospective studies suggest
that enterovirus exposure in childhood and even in utero may
increase the risk of IDDM (7, 8). In a cohort study carried out
in Finland, enterovirus infections were temporally associated
with the appearance or increases in circulating islet cell au-
toantibodies (ICA) (8). Furthermore, the children who converted to ICA seropositivity during an enterovirus infection
more often had the high risk human leukocyte antigen-DQB1
genotype than subjects who remained ICA negative (9).
Enterovirus infection in man usually starts from respiratory or gastrointestinal mucosa, spreads through the lymphatics to the circulation, and after a brief viraemic phase
may establish secondary replication sites in specific tissues
and organs. Some viruses have a specificity for anterior horn
cells of the spinal cord, whereas others have a propensity for
skeletal muscle or the heart (12). It is possible that some
enterovirus infections can reach the pancreatic islets and
bring about damage to the insulin-producing b-cells. In fact,
evidence of insulitis and b-cell damage has been seen in
histological examination of the pancreas from children dying
of overwhelming coxsackievirus B (CBV) infections (13, 14).
In two human cases, CBVs have been isolated from children
with acute-onset diabetes, and the isolates were also shown
to cause diabetes when injected into mice (1, 15). In a mouse
model it has been shown that the prototype strains of CBVs,
which initially failed to produce diabetes in mice, could be
made diabetogenic by passaging the virus either in mouse
pancreas or in cultures enriched in mouse b-cells (16, 17). The
mouse b-cell-adapted CBV-4/J.V.B. was also capable of producing transient diabetes in nonhuman primates (18). Furthermore, cultured human b-cells are susceptible to the diabetogenic isolate E2 of CBV-4 and CBV-3 (1, 19, 20).
Received June 16, 1999. Revision received August 30, 1999. Accepted
September 2, 1999.
Address all correspondence and requests for reprints to: Dr. Merja
Roivainen, Enterovirus Laboratory, National Public Health Institute,
Mannerheimintie 166, FIN-00300 Helsinki, Finland. E-mail: merja.
[email protected].
* This work was supported by grants from the Foundation for Diabetes Research in Finland, the Academy of Finland, the Sigrid Jusélius
Foundation, the Research Fund of the Helsinki University Central Hospital, the Juvenile Diabetes Foundation International (Grant 198202), and
the European Community (Contract BMH4-CT98 –3952). Preparation of
human islet cells by the Central Unit of the b-Cell Transplant is supported by grants from the Juvenile Diabetes Foundation International
and by a Shared Cost Action of the Medical and Health Research of the
European Community (Contract BMH-CT 95–1561).
† Recipient of a Juvenile Diabetes Foundation International Career
Development Award.
432
ENTEROVIRUS INFECTION IN PANCREATIC b-CELLS
Enteroviruses might induce or accelerate the process,
eventually resulting in clinical IDDM through several mechanisms. Pancreatic b-cells could be directly destroyed by
virus-induced cytolysis. Alternatively, a less aggressive enterovirus infection could cause an inflammatory reaction in
the islets, which could damage b-cells (21, 22) or lead to the
initiation of a b-cell-targeted autoimmune process. Homologous regions in enteroviral and islet cell proteins have also
prompted suggestions that enterovirus-induced b-cell damage might be based on molecular mimicry (23, 24).
Previous studies with isolated pancreatic islets have revealed that human b-cells are much more resistant against
toxins and cytokine-induced damage than rodent b-cells (25,
26). This underlines the importance of using human b-cells
for the detection of clinically relevant effects of enteroviruses.
Assuming that infection of b-cells is relevant to the diabetogenic effects of enterovirus infections, it is important to
know whether there are differences between enteroviruses in
their capacity to affect b-cells. For this purpose, we investigated the patterns and consequences of infection by several
coxsackievirus prototypes in human b-cells. Dynamic insulin
release was studied using islet perifusion to detect even
subtle adverse effects on b-cell function. Furthermore, we
have explored the mechanism of coxsackievirus-induced
b-cell death.
Materials and Methods
Human islets
Human pancreatic islets were isolated and purified (27) in Brussels
at the Central Unit of the b-Cell Transplant (coordinator: Prof. D. Pipeleers) and sent to Helsinki as free floating islets after 3–10 days of culture
in serum-free medium (Ham’s F-10 containing 1% BSA, penicillin, and
streptomycin). In our laboratory islets were cultured in the same medium supplemented with 25 mmol/L HEPES, pH 7.4 (incubation medium), with medium changes twice a week. The mean proportion of
b-cells in the human islet preparations was 56 6 14% (mean 6 sd; n 5
15), as determined at b-Cell Transplant (Brussels, Belgium) (27).
433
methanol for 15 min at 220 C. After washing [three times with phosphate-buffered saline (PBS)], they were double stained overnight at
room temperature with enterovirus-specific polyclonal rabbit antiserum
(1:300; KTL-510) (29) and insulin-specific polyclonal sheep antiserum (30
mg/mL; PC059, The Binding Site, Birmingham,UK). Visualization was
achieved by fluorescein isothiocyanate (FITC)-conjugated (711– 095-152,
Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and lissamine rhodamine (LRSC)-conjugated (713– 085-147, Jackson ImmunoResearch Laboratories, Inc.) antispecies sera. Photographs were taken
using a Carl Zeiss Axiophot fluorescence microscope (New York, NY)
and Fuji Photo Film Co., Ltd. Super G Plus 100 film (Tokyo, Japan).
DNA and insulin content of cells
For measurements of DNA and insulin content, islet cells were homogenized ultrasonically in distilled water. DNA was measured from
dried samples fluorometrically based on diaminobenzoic acid-induced
fluorescence (30). Insulin was measured with a commercial solid phase
insulin RIA kit (Diagnostic Products, Los Angeles, CA) after overnight
extraction with acid-ethanol as described previously (31).
Cell viability
The viability of islet cells after infection was measured using the
live/dead cell assay kit (L-3224, Molecular Probes, Inc., Leiden, The
Netherlands). The assay is based on the simultaneous determination of
live and dead cells with two fluorescent probes. Live cells are stained
green by calcein due to their esterase activity, and nuclei of dead cells
are stained red by ethidium homodimer-1. According to manufacturer’s
instructions, islets harvested at different time points were incubated
with the labeling solution for 30 min at room temperature in the dark,
cytocentrifuged onto glass slides, and analyzed with a Carl Zeiss Axiophot fluorescence microscope.
Cell type-specific apoptosis
Free floating islets were infected with apparent high multiplicity
(multiplicity of infection, 30 –100) of different virus preparations. After
adsorption for 1 h at 36 C, the inoculum virus was removed, and the cells
were washed twice with Hanks’ Balanced Salt Solution supplemented
with 20 mmol/L HEPES, pH 7.4. Incubation medium was then added
to all cultures, including uninfected controls, and the virus was allowed
to replicate at 36 C. Samples of suspended islets taken at different
intervals were frozen and thawed three times to release the virus, clarified by low speed centrifugation, and assayed for total infectivity using
end-point dilutions in microwell cultures of GMK cells. Cytopathic
effects were read on day 6 by microscopy, and 50% tissue culture infectious dose titers were calculated using the Kärber formula (28).
Cytocentrifuge preparations were obtained from the infected human
islet cells. The samples were fixed in 4% paraformaldehyde (for 30 min
at room temperature) and permeabilized by 1% sodium citrate-1% Triton X-100 (for 2 min on ice). To detect apoptosis, the cells were then
stained using the terminal dideoxynucleotidyltransferase (Tdt)-mediated digoxigenin-dideoxy (dd)-UTP nick end labeling (TUNEL) procedure. Reagents were purchased from Roche Molecular Biochemicals
(Mannheim, Germany). The preparations were preincubated in 5
mmol/L CaCl2-TdT buffer for 10 min and then DNA nick end labeled
by Tdt for 60 min at 37 C (5 mmol/L CaCl2, 5 mmol/L Tdt buffer, 0.23
mmol/L ddATP, 0.13 mmol/L dig-ddUTP, and 0.58 U/mL Tdt). To
detect the labeled cells, the samples were first blocked by 2% blocking
reagent in 150 mmol/L NaCl and 100 mmol/L Tris-HCl and were then
treated with horseradish peroxidase-conjugated antidigoxigenin Fab,
0.19 U/mL in blocking buffer, for 60 min in 37 C. The apoptotic nuclei
were visualized by a peroxidase dye (nitro blue tetrazolium/5-bromo4-chloro-3-indoyl-phosphate solution in 67% dimethylsulfoxide) for up
to 15 min. For double staining, the TUNEL procedure was followed by
insulin immunocytochemistry. The preparations were washed three
times with PBS and then blocked in 3% goat serum for 60 min at room
temperature. The antibody treatment (1:500 guinea pig antiporcine insulin antibody in 3% goat serum) was performed overnight at room
temperature. After rinsing several times with PBS, the samples were
incubated for 30 min at room temperature with biotinylated goat antirabbit IgG (Zymed Laboratories, Inc.), rinsed, and incubated with peroxidase-conjugated streptavidin (Zymed Laboratories, Inc., San Francisco, CA), diluted 1:100 in PBS. Finally, the insulin signal was developed
with AEC. Light counterstaining was performed with hematoxylin. A
similar procedure without Tdt treatment was used as a negative control
for every series of preparations. The result was quantified by counting
the numbers of all insulin-positive cells in the preparations, which were
scored as either TUNEL positive or TUNEL negative.
Immunocytochemistry
Electron microscopy
Samples of infected and uninfected islets were harvested at different
intervals on glass slides using a cytocentrifuge and fixed with cold
Cell pellets were fixed in glutaraldehyde followed by osmium tetroxide, dehydrated in graded ethanols, and embedded in Spurr resin.
Viruses
Prototype strains of enteroviruses (CBV-3/Nancy, CBV-4/J.V.B.,
CBV-5/Faulkner, and CAV-9/Griggs) were obtained from American
Type Culture Collection (Manassas, VA). The diabetes-associated strain
CBV-4-E2 was obtained from Dr. J.-W. Yoon (1). All viruses were passaged in GMK cells, a continuous cell line of green monkey kidney
origin. The identity of all enterovirus preparations used was confirmed
using a plaque neutralization assay with type-specific antisera.
Replication of viruses
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Ultrathin sections were counterstained with uranyl acetate and lead
citrate before examination under the electron microscope.
Nitrite concentration in culture medium
The method used for nitrite measurements is slightly modified from
that described previously (32). One hundred microliters of culture medium were incubated with 10 mL reagent (10% sulfanilamide in 50%
phosphoric acid and 1% naphthyl ethylenediamine dihydrochloride) for
2 min at 60 C, and nitric oxide (NO) was determined as nitrite from the
absorbance at 550 nm, using sodium nitrite as standard.
Inducible NO synthase (iNOS) messenger ribonucleic acid
(mRNA) in cells
Extraction of mRNA from infected and uninfected cells (;0.8 3 106
b-cells/assay) was carried out using a commercial isolation procedure
(Oligotex Direct mRNA Micro Protocol, QIAGEN, Valencia, CA). RTPCR for human iNOS and for the housekeeping gene glyceraldehyde3-phosphate dehydrogenase (GAPDH) were performed as previously
described (33) using 31 and 34 cycles for GAPDH and iNOS, respectively. The ethidium bromide-stained agarose gels were photographed
under UV transillumination using a Kodak Digital Science DC40 camera
(Eastman Kodak, Rochester, NY), and the PCR band intensities on the
image were quantified by Biomax 1D Image Analysis Software (Kodak)
and expressed in pixel intensities (optical densities). All values for iNOS
were corrected for the respective GAPDH values.
Insulin secretion
Insulin release in response to glucose and glucose plus theophyline
was studied separately by perifusion as described previously (34). The
same number of islets was originally included in each assay. Briefly, after
taking samples for insulin content and DNA measurements, control
islets and islets infected for 1–7 days were loaded in perifusion chambers
in Krebs-Ringer bicarbonate buffer supplemented with 20 mmol/L
HEPES (pH 7.35) and 0.2% BSA. The buffer was pumped through the
chambers at a flow rate of 0.25 mL/min. After a 60-min stabilizing period
in low glucose (1.67 mmol/L), fractions were collected every 4 min
(sample volume, 1 mL). The glucose concentration was changed to 16.7
mmol/L at fraction 3. Due to the dead space of the system (3.5 mL), the
actual measured glucose concentration of the effluate reached the maximum at fraction 6. The first phase peak response to glucose was mea-
FIG. 1. Replication of enteroviruses in
adult human islets. Parallel aliquots of
free floating islets were infected with
apparent high multiplicity (30 –100
plaque-forming units/well) of CBV-4E2, CBV-3, CBV-4/J.V.B., CBV-5, and
CAV-9. After a 1-h adsorption period
the inoculum virus was removed, islets
were washed twice, and culture medium was added. Samples taken at different intervals were assayed for total
infectivity. The results are shown as the
mean 6 95% confidence intervals of
three experiments.
sured from the insulin concentration in fraction 6. The second phase
response was calculated from the mean insulin content in fractions
10 –14 while maintaining the high glucose concentration. The cells were
finally stimulated with a mixture of 16.7 mmol/L glucose and 10
mmol/L theophyline (Sigma, St. Louis, MO) during fractions 13–15
(reaching the effluate in fractions 16 –18), after which the basal buffer
(1.67 mmol/L glucose) was used during the final final fractions. Five or
six perifusion lines were run in parallel using a multichannel perifusion
apparatus (Brandel, Gaithersburg, MD).
Statistical methods
Differences between groups were tested with StatView 4.1 software
for the Macintosh (Abacus Concepts, Berkeley, CA), using one-way
ANOVA followed by Fischer’s protected least significant difference test,
taking 95% level as the limit of significance.
Results
Cultures of human b-cells were infected with prototype
strains of CBV-3, CBV-4/J.V.B., CBV-5, and CAV-9 and the
diabetogenic strain, CBV-4-E2. Virus replication was studied
by measuring the infectivity of samples collected at different
intervals. The results showed that all viruses replicated well
in all human islet preparations tested (Fig. 1).
In all CBV-infected cultures the first morphological
changes were seen by 1–2 days after infection, whereas CAV9-infected cells seemed to be virtually intact even at 7 days
after infection. Cells infected with CBVs first became more
rounded and then they detached from the islet into the medium. As a result, the size of the islets decreased gradually
during infection. After longer incubation, the virus-induced
cytopathic effect became more pronounced, but some islet
cells remained intact over the entire observation period. Infection-induced damage was also evident by the live-dead
cell assay. At 4.5 days after infection the whole islet was
covered by dead cells, as shown in Fig. 2 for the prototype
strain of CBV-4/J.V.B.
ENTEROVIRUS INFECTION IN PANCREATIC b-CELLS
435
FIG. 2. Effect of enterovirus infection
on human islet cell viability. CBV-4/
J.V.B.-infected and uninfected control
islets were harvested at 2 and 4.5 days
after infection and stained with the live/
dead cell assay kit. Live cells are
stained green by calcein due to their esterase activity, whereas red fluorescence is induced in the nuclei of dead
cells by ethidium homodimer-1. Cofluorescence from adjacent overlapping
cells results in a yellow color. Peripheral
islet cells are dying at 4.5 days of infection. The figure is representative of nine
similar experiments.
To confirm that replication of enteroviruses took place in
insulin-producing b-cells, we used a double antibody immunofluorescence technique with insulin and virus-specific
antisera. Binding of antibodies was visualized by FITC- and
LRSC-labeled conjugates. Virus-infected insulin-containing
cells were readily identified by their yellow or orange color
(Fig. 3).
Electron microscopy of human islets revealed that after 2
days of infection, about 40% of CBV-5 infected cells had
undergone morphological changes characteristic of pyknosis, i.e. highly distorted and wrinkled nuclei that often were
displaced to the cellular periphery. These nuclei had a crescent shape, with deep indentations and highly condensed
chromatin (Fig. 4A). There were no signs of nuclear or cytoplasmic fragmentation or of crescent-shaped chromatin
condensations, both known to be characteristic of apoptosis.
Plasma membranes were intact in these cells. A similar morphology was not found in control cells. One week after infection, the majority of cells showed characteristics of secondary necrosis. In the pyknotic cells, polyhedral virus
particles measuring approximately 30 nm could be observed
in the cytoplasm (Fig. 4B).
Insulin release and content
As expected, uninfected adult human b-cells responded to
glucose with a biphasic response of insulin release, with the
first phase peak (4.1 6 0.9-fold over the basal level) occurring
in the first poststimulatory fraction, followed by a prolonged
second phase (2.6 6 0.5-fold over the basal level). Finally, the
glucose response was further potentiated by 10 mmol/L
theophyline (6.9 6 1.2-fold).
CBV infection induced a readily detectable perturbance in
insulin release. The results of experiments performed 7 days
after infection are summarized in Fig. 5. The most deleterious
viruses were CBV-3 and CBV-5. In all experiments the glucose responses of islets infected with these viruses were
significantly decreased within 1 week (Fig. 5A). The susceptibility of the cells to CBV-4 strains was somewhat more
variable. As a result, only CBV-3 and CBV-5 significantly
affected the first and second phase responses to glucose. Both
strains of CBV4 also impaired the response to theophyline
plus glucose. Unlike CBV-infected islets, CAV-9-infected
cells responded well to both stimuli at 1 week after infection
(Fig. 5A).
Insulin content was studied from the aliquots of samples
harvested at different time points for perifusion. Results
obtained on day 7 are summarized in Fig. 5B. At this time,
the insulin content per cellular DNA of islets infected by
CBV4-E2 and CBV-5 had decreased by about 40%, and that
of those infected by CBV-3 had decreased by 65% compared
with that in uninfected control islets. The effect of CBV-4/
J.V.B. did not reach statistical significance. Analogous with
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FIG. 3. Enterovirus infection in insulin-producing human b-cells. A, Uninfected control islets and islets infected with CBV-4-E2, CBV-4/J.V.B.,
CBV-5, or CAV-9 and harvested at 1–2 days after infection. B, Uninfected and CBV-3-infected islets harvested 1 day after infection. Harvested
cells were cytocentrifuged on glass slides, fixed with cold methanol, and double stained with enterovirus-specific rabbit antiserum and
insulin-specific sheep antiserum. Visualization was performed by FITC-antirabbit (green, for virus antigens) and LRSC- antisheep (red, for
insulin) conjugates. Yellow-orange double fluorescence is seen after infection with all viruses, indicating that the viruses have effectively infected
the b-cells. A and B are representative of nine and three similar experiments, respectively. CBV-3-infected islets together with uninfected
controls were studied in separate experiments and analyzed by confocal microscopy.
the insulin release data, the insulin content of CAV-9-infected cells remained intact.
DNA fragmentation
Based on TUNEL, only a minority of the infected cells
became apoptotic (Fig. 6). However, at 2 days after infection,
DNA fragmentation in the nuclei of b-cells was significantly
increased in CBV-5-infected cells (5.9 6 1.1%; P , 0.001) and
tended to be increased also in CBV-4-infected cells (3.9 6
0.5%; P 5 0.06) compared with that in noninfected controls
(2.1 6 0.3%) and CAV-9-infected cells (2.6 6 0.5%; Fig. 6B).
There were no significant differences in the numbers of
TUNEL-positive cells after 7 days, when the majority of
CBV-infected cells had died or had pyknotic nuclei as determined by electron microscopy.
iNOS mRNA expression
The possibility that NO mediates infection-induced b-cell
impairment was studied by measuring iNOS mRNA expression and nitrite accumulation in the culture medium. The
cellular content of iNOS mRNA was determined by semiquantitative RT-PCR of mRNA isolated from CBV-5-infected
cells and controls. As a positive control, the islets were stimulated for 24 h with interferon-g (1000 U/mL) and interleukin-1b (30 U/mL). One day of CBV-5 infection did not modify iNOS expression, whereas culture in the presence of the
cytokines induced a 5-fold increase in human islet iNOS
expression (Fig. 7). A 2-day viral infection also failed to increase
iNOS mRNA expression (data not shown), and there was no
consistent increase in medium nitrite accumulation after 2–10
days of infection with CBV-5 or CBV-3 (data not shown).
ENTEROVIRUS INFECTION IN PANCREATIC b-CELLS
FIG. 4. Electron microscopy of infected human islets. A, Electron
micrograph of CBV-5-infected cells (2 days; magnification, 34000)
showing a b-cell (left) and an a-cell (right). Both cells show morphological evidence of pyknosis, i.e. distorted nucleus with condensed
chromatin. B, Higher magnification (340 000) of a CBV-5-infected cell
showing virus particles in the cytoplasm. The arrowheads point to
virus particles.
Discussion
The present study shows that in addition to the diabetogenic strain E2 of CBV-4, the prototype strains of CBV-3,
CBV-4, CBV-5, and CAV-9 are able to infect insulin-producing b-cells in primary adult human islets. The insulin release
responses to secretagogues were markedly impaired in CBVinfected b-cells, even when their insulin content was only
marginally decreased. These effects were partly due to b-cell
437
lysis, but the remaining b-cells also appeared to be functionally impaired. CBV-3 and CBV-5 induce the most severe
signals of b-cell dysfunction and damage, whereas CAV-9
infection did not cause any apparent adverse effects on b-cell
function during the 7-day follow-up.
Enteroviruses are classically associated with lytic infections, but they can also establish noncytolytic or chronic
infections (22, 35). Recently two types of death mechanisms
were reported for one enterovirus, poliovirus type 1 (36). It
was shown that productive virus infection in HeLa cells
results in pyknosis characterized by highly distorted nuclei
with condensed, but intact, chromatin, whereas conditions
restricting viral production are associated with typical apoptosis. Our ultrastructural observations demonstrated that
CBV-5-infected cells died by the process of pyknosis and not
by apoptosis. Only a minority of the infected cells became
apoptotic, as evidenced by nuclear morphology and increased in situ DNA end labeling. This suggests that apoptosis may not have a major role in CBV-5-induced cell death
during a productive infection.
Nitric oxide may be a mediator of b-cell death in diabetes
mellitus (reviewed in Ref. 37), and there is evidence that viral
infection leads to NO production by different cell types (38,
39). In the present experiments there was no direct induction
of iNOS expression and nitrite production by CBV-5 infection, suggesting that virally induced b-cell death was not
mediated by NO. It cannot, however, be excluded that viral
infection in vivo, accompanied by local production of cytokines by infiltrating immune cells, will lead to islet NO
production.
Unlike CBVs, CAV-9 appeared to cause a noncytolytic
infection. It replicated well in human b-cells, and the infected
islets still responded like uninfected control cells at 1 week
after infection. The effects of CAV-9 were studied because it
is genetically closely related to the CBVs (40), but its biological effects are distinct. In newborn mice, for example, it
affects only skeletal muscle, whereas CBVs are capable of
affecting several other organs as well (41). Most importantly,
CAV-9 was one of the enteroviruses found to be temporally
associated with increases in the levels of islet cell antibodies
in prediabetic children (42). Based on our findings, persistent CAV-9 infection of the b-cells could be one mechanism linking enterovirus infections with b-cell targeted
autoimmunity.
Some heterogeneity was apparent in the susceptibility of
the b-cells to the effects of enteroviruses even within a single
experiment. Although some b-cells were lysed, neighboring
b-cells remained virtually intact in the CBV-infected cultures. This is in accordance with the well known situation at
the onset of diabetes, when some islets may still be intact
while in others only noninsulin-producing cells remain (reviewed in Ref. 43). Similar observations have also been reported previously with infection of cultured human islets
with the diabetogenic strain E2 of CBV-4 (1). This could
reflect the metabolic heterogeneity of the b-cells. Thus, only
a proportion of b-cells becomes metabolically activated during glucose stimulation (44), and this active b-cell subpopulation is preferentially inhibited by the cytokine interleu-
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FIG. 5. A, Stimulated release of insulin
in adult human islet cells in perifusion
experiments at 7 days after enterovirus
infection. Results are shown for the cumulative data obtained with nine human islet preparations. Insulin release
is expressed as the stimulation index
(stimulated/basal level) in response to
16.7 mmol/L glucose, first (GLUG 1)
and second (GLUG 2) phases separately
(see Materials and Methods for their
definition), and to glucose plus 10
mmol/L theophyline (G1T). B, Insulin
content per cellular DNA of the human
islets harvested at 7 days after infection, expressed as relative changes from
the uninfected control. The number of
observations is indicated at the bottom
of the columns. *, P , 0.05; **, P , 0.01
(compared with the uninfected control
cells).
kin-1b (45). Whether the metabolically active b-cells are also
the ones most severely affected by enteroviruses remains
unknown at present.
The induction of virus-induced diabetes in mice is known
to depend on the genetic background of the host and the
passage history of the virus (46, 47). The human isolate E2 of
CBV-4 can induce a diabetes-like syndrome in mice. The
prototype strain of CBV-4/J.V.B. is able to replicate in murine
pancreatic b-cells, but it does not cause cell lysis or produce
glucose abnormalities (17, 47). However, diabetogenicity of
this virus strain can be enhanced by passaging it either in vivo
in mouse pancreas or in b-cell cultures (16, 17). In contrast to
murine pancreatic b-cells, human adult b-cells were found
here to be susceptible to the cytolytic effects of the prototype
ENTEROVIRUS INFECTION IN PANCREATIC b-CELLS
439
FIG. 6. TUNEL staining for the simultaneous in situ detection of DNA fragmentation (black nuclear staining), and insulin (red-brown
cytoplasmic staining) in uninfected human islets (A) and 2 days after infection with CBV-5 (B). Arrows indicate TUNEL-positive b-cells. C,
Quantitation of the apoptotic b-cells at 2 days of infection from four to six separate experiments (mean 6 SEM). **, P , 0.01 compared with
control.
FIG. 7. Semiquantitative RT-PCR for the detection of iNOS mRNA.
Islets were harvested after 24 h of infection or cytokine treatment.
Lane 1, Human islets treated with IL-1b (50 U/mL) and interferon-g
(1000 U/mL); lane 2, uninfected islets; lane 3, islets infected with
CBV-5. The results of densitometric quantitation of iNOS/GAPDH
expression are shown at the bottom. The figure is representative of
three similar experiments.
strains of CBV-4/J.V.B. and CBV-5. The reasons for the observed differences between species are not known at present.
The most deleterious viruses in adult human islets were
CBV-3 and CBV-5. CBV-5 is known to occur as explosive
epidemics (11). Interestingly, increased incidence of IDDM
has been reported after epidemics of CBV-5 (10, 11), and
CBV-5 epidemics occur frequently in Finland, where the
incidence of IDDM is the highest in the world (48).
Although some potentially diabetogenic strains of enteroviruses have been described (1, 15), there is no strong evidence to suggest that the putative diabetogenic property
would be restricted to a single or even a few strains or
serotypes only. It is possible that when infecting a genetically
susceptible individual, several different serotypes or perhaps
even all enteroviruses could be diabetogenic. The large number of different enterovirus serotypes makes identification of
the most pathogenic serotypes an important, but demanding,
task. The screening process could be simplified significantly
by standardized experiments with primary islet cultures, as
presently presented.
In conclusion, we have shown that several prototype
strains of enteroviruses infect human b-cells. The responses
of infection were different from those previously reported in
rodent b-cells. In human b-cells, CBV typically cause a lytic
infection, characterized by nuclear pyknosis, but only some
of the b-cells are immediately killed. The functional capacity
of the remaining b-cells is also deteriorated. Coxsackievirus
A9 represents a noncytolytic type of infection in the b-cell.
Such an infection could theoretically lead to the initiation or
exaggeration of b-cell-targeted autoimmunity.
Acknowledgments
We thank Prof. J.-W. Yoon for the diabetogenic E2 strain of CBV-4
used in the study, Ms. Mervi Eskelinen for skillful technical assistance,
and the personnel of the b-Cell Transplant (Brussels, Belgium) for the
preparation of human islet cells.
440
ROIVAINEN ET AL.
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