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Original Studies |
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
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.roivainen{at}ktl.fi
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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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 ß-cells. In fact, evidence of insulitis and ß-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 ß-cells (16, 17). The mouse ß-cell-adapted CBV-4/J.V.B. was also capable of producing transient diabetes in nonhuman primates (18). Furthermore, cultured human ß-cells are susceptible to the diabetogenic isolate E2 of CBV-4 and CBV-3 (1, 19, 20).
Enteroviruses might induce or accelerate the process, eventually resulting in clinical IDDM through several mechanisms. Pancreatic ß-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 ß-cells (21, 22) or lead to the initiation of a ß-cell-targeted autoimmune process. Homologous regions in enteroviral and islet cell proteins have also prompted suggestions that enterovirus-induced ß-cell damage might be based on molecular mimicry (23, 24).
Previous studies with isolated pancreatic islets have revealed that human ß-cells are much more resistant against toxins and cytokine-induced damage than rodent ß-cells (25, 26). This underlines the importance of using human ß-cells for the detection of clinically relevant effects of enteroviruses. Assuming that infection of ß-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 ß-cells. For this purpose, we investigated the patterns and consequences of infection by several coxsackievirus prototypes in human ß-cells. Dynamic insulin release was studied using islet perifusion to detect even subtle adverse effects on ß-cell function. Furthermore, we have explored the mechanism of coxsackievirus-induced ß-cell death.
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Materials and Methods |
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Human pancreatic islets were isolated and purified (27) in Brussels at the Central Unit of the ß-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 ß-cells in the human islet preparations was 56 ± 14% (mean ± SD; n = 15), as determined at ß-Cell Transplant (Brussels, Belgium) (27).
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
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).
Immunocytochemistry
Samples of infected and uninfected islets were harvested at different intervals on glass slides using a cytocentrifuge and fixed with cold methanol for 15 min at -20 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 µg/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
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/µL 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-bromo-4-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.
Electron microscopy
Cell pellets were fixed in glutaraldehyde followed by osmium tetroxide, dehydrated in graded ethanols, and embedded in Spurr resin. 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 µL 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 x 106 ß-cells/assay) was carried
out using a commercial isolation procedure (Oligotex Direct mRNA Micro
Protocol, QIAGEN, Valencia, CA). RT-PCR for human iNOS and
for the housekeeping gene glyceraldehyde-3-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 measured 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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for the prototype strain of
CBV-4/J.V.B.
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).
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). 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).
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As expected, uninfected adult human ß-cells responded to glucose with a biphasic response of insulin release, with the first phase peak (4.1 ± 0.9-fold over the basal level) occurring in the first poststimulatory fraction, followed by a prolonged second phase (2.6 ± 0.5-fold over the basal level). Finally, the glucose response was further potentiated by 10 mmol/L theophyline (6.9 ± 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).
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. 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 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 ß-cells was
significantly increased in CBV-5-infected cells (5.9 ± 1.1%;
P < 0.001) and tended to be increased also in
CBV-4-infected cells (3.9 ± 0.5%; P = 0.06)
compared with that in noninfected controls (2.1 ± 0.3%) and
CAV-9-infected cells (2.6 ± 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.
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The possibility that NO mediates infection-induced ß-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-
(1000 U/mL) and
interleukin-1ß (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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 ß-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 ß-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 ß-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 ß-cells could be one mechanism linking enterovirus infections with ß-cell targeted autoimmunity.
Some heterogeneity was apparent in the susceptibility of the ß-cells to the effects of enteroviruses even within a single experiment. Although some ß-cells were lysed, neighboring ß-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 ß-cells. Thus, only a proportion of ß-cells becomes metabolically activated during glucose stimulation (44), and this active ß-cell subpopulation is preferentially inhibited by the cytokine interleukin-1ß (45). Whether the metabolically active ß-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 ß-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 ß-cell cultures (16, 17). In contrast to murine pancreatic ß-cells, human adult ß-cells were found here to be susceptible to the cytolytic effects of the prototype 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 ß-cells. The responses of infection were different from those previously reported in rodent ß-cells. In human ß-cells, CBV typically cause a lytic infection, characterized by nuclear pyknosis, but only some of the ß-cells are immediately killed. The functional capacity of the remaining ß-cells is also deteriorated. Coxsackievirus A9 represents a noncytolytic type of infection in the ß-cell. Such an infection could theoretically lead to the initiation or exaggeration of ß-cell-targeted autoimmunity.
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Acknowledgments |
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Footnotes |
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2 Recipient of a Juvenile Diabetes Foundation International Career
Development Award.
Received June 16, 1999.
Revised August 30, 1999.
Accepted September 2, 1999.
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-cell (right). Both cells show morphological evidence
of pyknosis, i.e. distorted nucleus with condensed
chromatin. B, Higher magnification (x40 000) of a CBV-5-infected cell
showing virus particles in the cytoplasm. The arrowheads
point to virus particles.
