Mechanisms of Coxsackievirus-Induced Damage to Human Pancreaticβ -Cells 1

Mechanisms of Coxsackievirus-Induced Damage toHuman Pancreatic b-Cells*

MERJA ROIVAINEN, SUVI RASILAINEN, PETRI YLIPAASTO, RIIKKA NISSINEN,JARKKO USTINOV, LUC BOUWENS, DECIO 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 andAdolescents (T.O.), University of Helsinki, FIN-00014 Helsinki, Finland; and the Diabetes ResearchCenter, Vrije Universiteit Brussel (L.B., D.L.E.), B-1090 Brussels, Belgium

ABSTRACTEnteroviruses may be involved in the pathogenesis of insulin-

dependent diabetes mellitus, either through direct b-cell infection oras triggers of autoimmunity. In the present study we investigated thepatterns of infection in adult human islet cell preparations (consistingof 56 6 14% b-cells) by several coxsackieviruses. The cells were in-fected with prototype strains of coxsackievirus B (CBV) 3, 4, and 5 aswell as coxsackievirus A9 (CAV-9). The previously characterized di-abetogenic strain of coxsackievirus B4 (CBV-4-E2) was used as areference. All viruses replicated well in b-cells, but only CBVs causedcell death. One week after infection, the insulin response of the b-cellsto glucose or glucose plus theophyline was most severely impaired byCBV-3 and CBV-5 infections. CBV-4 also caused significant func-tional impairment, whereas CAV-9-infected cells responded like un-infected controls. After 2 days of infection, about 40% of CBV-5-infected cells had undergone morphological changes characteristic of

pyknosis, i.e. highly distorted nuclei with condensed but intact chro-matin. Both mitochondria and plasma membrane were intact in thesecells. DNA fragmentation was found in 5.9 6 1.1% of CBV-5-infectedb-cell nuclei (2.1 6 0.3% in controls; P , 0.01). CAV-9 infection didnot induce DNA fragmentation. One week after infection the majorityof infected cells showed characteristics of secondary necrosis. Mediumnitrite and inducible nitric oxide synthase messenger ribonucleic acidlevels were not significantly up-regulated by CBV infection. Theseresults suggest that several enteroviruses may infect human b-cells.The infection may result in functional impairment or death of theb-cell or may have no apparent immediate adverse effects, as shownhere for CAV-9. Coxsackie B viruses cause functional impairment andb-cell death characterized by nuclear pyknosis. Apoptosis appears toplay a minor role during a productive CBV infection in b-cells. (J ClinEndocrinol Metab 85: 432–440, 2000)

INSULIN-DEPENDENT diabetes mellitus (IDDM) iscaused by progressive destruction of pancreatic b-cells,

resulting in insulin deficiency. Several lines of epidemiolog-ical evidence suggest that enterovirus infections, especiallythose due to the group B of coxsackieviruses may have a rolein the etiology of IDDM (1–9). According to some studies,outbreaks of IDDM may be associated with enterovirus ep-idemics (10, 11). Results from prospective studies suggestthat enterovirus exposure in childhood and even in utero mayincrease the risk of IDDM (7, 8). In a cohort study carried outin Finland, enterovirus infections were temporally associatedwith the appearance or increases in circulating islet cell au-

toantibodies (ICA) (8). Furthermore, the children who con-verted to ICA seropositivity during an enterovirus infectionmore often had the high risk human leukocyte antigen-DQB1genotype than subjects who remained ICA negative (9).

Enterovirus infection in man usually starts from respira-tory or gastrointestinal mucosa, spreads through the lym-phatics to the circulation, and after a brief viraemic phasemay establish secondary replication sites in specific tissuesand organs. Some viruses have a specificity for anterior horncells of the spinal cord, whereas others have a propensity forskeletal muscle or the heart (12). It is possible that someenterovirus infections can reach the pancreatic islets andbring about damage to the insulin-producing b-cells. In fact,evidence of insulitis and b-cell damage has been seen inhistological examination of the pancreas from children dyingof overwhelming coxsackievirus B (CBV) infections (13, 14).In two human cases, CBVs have been isolated from childrenwith acute-onset diabetes, and the isolates were also shownto cause diabetes when injected into mice (1, 15). In a mousemodel it has been shown that the prototype strains of CBVs,which initially failed to produce diabetes in mice, could bemade diabetogenic by passaging the virus either in mousepancreas or in cultures enriched in mouse b-cells (16, 17). Themouse b-cell-adapted CBV-4/J.V.B. was also capable of pro-ducing transient diabetes in nonhuman primates (18). Fur-thermore, cultured human b-cells are susceptible to the di-abetogenic isolate E2 of CBV-4 and CBV-3 (1, 19, 20).

Received June 16, 1999. Revision received August 30, 1999. AcceptedSeptember 2, 1999.

Address all correspondence and requests for reprints to: Dr. MerjaRoivainen, Enterovirus Laboratory, National Public Health Institute,Mannerheimintie 166, FIN-00300 Helsinki, Finland. E-mail: [email protected].

* This work was supported by grants from the Foundation for Dia-betes Research in Finland, the Academy of Finland, the Sigrid JuseliusFoundation, the Research Fund of the Helsinki University Central Hos-pital, the Juvenile Diabetes Foundation International (Grant 198202), andthe European Community (Contract BMH4-CT98–3952). Preparation ofhuman islet cells by the Central Unit of the b-Cell Transplant is sup-ported by grants from the Juvenile Diabetes Foundation Internationaland by a Shared Cost Action of the Medical and Health Research of theEuropean Community (Contract BMH-CT 95–1561).

† Recipient of a Juvenile Diabetes Foundation International CareerDevelopment Award.

0021-972X/00/$03.00/0 Vol. 85, No. 1The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A.Copyright © 2000 by The Endocrine Society

432

Enteroviruses might induce or accelerate the process,eventually resulting in clinical IDDM through several mech-anisms. Pancreatic b-cells could be directly destroyed byvirus-induced cytolysis. Alternatively, a less aggressive en-terovirus infection could cause an inflammatory reaction inthe islets, which could damage b-cells (21, 22) or lead to theinitiation of a b-cell-targeted autoimmune process. Homol-ogous regions in enteroviral and islet cell proteins have alsoprompted suggestions that enterovirus-induced b-cell dam-age might be based on molecular mimicry (23, 24).

Previous studies with isolated pancreatic islets have re-vealed that human b-cells are much more resistant againsttoxins and cytokine-induced damage than rodent b-cells (25,26). This underlines the importance of using human b-cellsfor the detection of clinically relevant effects of enteroviruses.Assuming that infection of b-cells is relevant to the diabe-togenic effects of enterovirus infections, it is important toknow whether there are differences between enteroviruses intheir capacity to affect b-cells. For this purpose, we investi-gated the patterns and consequences of infection by severalcoxsackievirus prototypes in human b-cells. Dynamic insulinrelease was studied using islet perifusion to detect evensubtle adverse effects on b-cell function. Furthermore, wehave explored the mechanism of coxsackievirus-inducedb-cell death.

Materials and MethodsHuman islets

Human pancreatic islets were isolated and purified (27) in Brusselsat the Central Unit of the b-Cell Transplant (coordinator: Prof. D. Pipel-eers) and sent to Helsinki as free floating islets after 3–10 days of culturein serum-free medium (Ham’s F-10 containing 1% BSA, penicillin, andstreptomycin). In our laboratory islets were cultured in the same me-dium supplemented with 25 mmol/L HEPES, pH 7.4 (incubation me-dium), with medium changes twice a week. The mean proportion ofb-cells in the human islet preparations was 56 6 14% (mean 6 sd; n 515), as determined at b-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 AmericanType Culture Collection (Manassas, VA). The diabetes-associated strainCBV-4-E2 was obtained from Dr. J.-W. Yoon (1). All viruses were pas-saged in GMK cells, a continuous cell line of green monkey kidneyorigin. The identity of all enterovirus preparations used was confirmedusing 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. Afteradsorption for 1 h at 36 C, the inoculum virus was removed, and the cellswere washed twice with Hanks’ Balanced Salt Solution supplementedwith 20 mmol/L HEPES, pH 7.4. Incubation medium was then addedto all cultures, including uninfected controls, and the virus was allowedto replicate at 36 C. Samples of suspended islets taken at differentintervals were frozen and thawed three times to release the virus, clar-ified by low speed centrifugation, and assayed for total infectivity usingend-point dilutions in microwell cultures of GMK cells. Cytopathiceffects were read on day 6 by microscopy, and 50% tissue culture in-fectious dose titers were calculated using the Karber formula (28).

Immunocytochemistry

Samples of infected and uninfected islets were harvested at differentintervals on glass slides using a cytocentrifuge and fixed with cold

methanol for 15 min at 220 C. After washing [three times with phos-phate-buffered saline (PBS)], they were double stained overnight atroom temperature with enterovirus-specific polyclonal rabbit antiserum(1:300; KTL-510) (29) and insulin-specific polyclonal sheep antiserum (30mg/mL; PC059, The Binding Site, Birmingham,UK). Visualization wasachieved by fluorescein isothiocyanate (FITC)-conjugated (711–095-152,Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and lis-samine rhodamine (LRSC)-conjugated (713–085-147, Jackson Immu-noResearch Laboratories, Inc.) antispecies sera. Photographs were takenusing 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 ho-mogenized ultrasonically in distilled water. DNA was measured fromdried samples fluorometrically based on diaminobenzoic acid-inducedfluorescence (30). Insulin was measured with a commercial solid phaseinsulin RIA kit (Diagnostic Products, Los Angeles, CA) after overnightextraction with acid-ethanol as described previously (31).

Cell viability

The viability of islet cells after infection was measured using thelive/dead cell assay kit (L-3224, Molecular Probes, Inc., Leiden, TheNetherlands). The assay is based on the simultaneous determination oflive and dead cells with two fluorescent probes. Live cells are stainedgreen by calcein due to their esterase activity, and nuclei of dead cellsare stained red by ethidium homodimer-1. According to manufacturer’sinstructions, islets harvested at different time points were incubatedwith the labeling solution for 30 min at room temperature in the dark,cytocentrifuged onto glass slides, and analyzed with a Carl Zeiss Ax-iophot fluorescence microscope.

Cell type-specific apoptosis

Cytocentrifuge preparations were obtained from the infected humanislet cells. The samples were fixed in 4% paraformaldehyde (for 30 minat room temperature) and permeabilized by 1% sodium citrate-1% Tri-ton X-100 (for 2 min on ice). To detect apoptosis, the cells were thenstained using the terminal dideoxynucleotidyltransferase (Tdt)-medi-ated digoxigenin-dideoxy (dd)-UTP nick end labeling (TUNEL) proce-dure. Reagents were purchased from Roche Molecular Biochemicals(Mannheim, Germany). The preparations were preincubated in 5mmol/L CaCl2-TdT buffer for 10 min and then DNA nick end labeledby Tdt for 60 min at 37 C (5 mmol/L CaCl2, 5 mmol/L Tdt buffer, 0.23mmol/L ddATP, 0.13 mmol/L dig-ddUTP, and 0.58 U/mL Tdt). Todetect the labeled cells, the samples were first blocked by 2% blockingreagent in 150 mmol/L NaCl and 100 mmol/L Tris-HCl and were thentreated with horseradish peroxidase-conjugated antidigoxigenin Fab,0.19 U/mL in blocking buffer, for 60 min in 37 C. The apoptotic nucleiwere visualized by a peroxidase dye (nitro blue tetrazolium/5-bromo-4-chloro-3-indoyl-phosphate solution in 67% dimethylsulfoxide) for upto 15 min. For double staining, the TUNEL procedure was followed byinsulin immunocytochemistry. The preparations were washed threetimes with PBS and then blocked in 3% goat serum for 60 min at roomtemperature. The antibody treatment (1:500 guinea pig antiporcine in-sulin antibody in 3% goat serum) was performed overnight at roomtemperature. After rinsing several times with PBS, the samples wereincubated for 30 min at room temperature with biotinylated goat anti-rabbit IgG (Zymed Laboratories, Inc.), rinsed, and incubated with per-oxidase-conjugated streptavidin (Zymed Laboratories, Inc., San Fran-cisco, CA), diluted 1:100 in PBS. Finally, the insulin signal was developedwith AEC. Light counterstaining was performed with hematoxylin. Asimilar procedure without Tdt treatment was used as a negative controlfor every series of preparations. The result was quantified by countingthe numbers of all insulin-positive cells in the preparations, which werescored as either TUNEL positive or TUNEL negative.

Electron microscopy

Cell pellets were fixed in glutaraldehyde followed by osmium tet-roxide, dehydrated in graded ethanols, and embedded in Spurr resin.

ENTEROVIRUS INFECTION IN PANCREATIC b-CELLS 433

Ultrathin sections were counterstained with uranyl acetate and leadcitrate before examination under the electron microscope.

Nitrite concentration in culture medium

The method used for nitrite measurements is slightly modified fromthat described previously (32). One hundred microliters of culture me-dium were incubated with 10 mL reagent (10% sulfanilamide in 50%phosphoric acid and 1% naphthyl ethylenediamine dihydrochloride) for2 min at 60 C, and nitric oxide (NO) was determined as nitrite from theabsorbance 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). RT-PCR for human iNOS and for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were performed as previouslydescribed (33) using 31 and 34 cycles for GAPDH and iNOS, respec-tively. The ethidium bromide-stained agarose gels were photographedunder UV transillumination using a Kodak Digital Science DC40 camera(Eastman Kodak, Rochester, NY), and the PCR band intensities on theimage were quantified by Biomax 1D Image Analysis Software (Kodak)and expressed in pixel intensities (optical densities). All values for iNOSwere corrected for the respective GAPDH values.

Insulin secretion

Insulin release in response to glucose and glucose plus theophylinewas studied separately by perifusion as described previously (34). Thesame number of islets was originally included in each assay. Briefly, aftertaking samples for insulin content and DNA measurements, controlislets and islets infected for 1–7 days were loaded in perifusion chambersin Krebs-Ringer bicarbonate buffer supplemented with 20 mmol/LHEPES (pH 7.35) and 0.2% BSA. The buffer was pumped through thechambers at a flow rate of 0.25 mL/min. After a 60-min stabilizing periodin low glucose (1.67 mmol/L), fractions were collected every 4 min(sample volume, 1 mL). The glucose concentration was changed to 16.7mmol/L at fraction 3. Due to the dead space of the system (3.5 mL), theactual measured glucose concentration of the effluate reached the max-imum at fraction 6. The first phase peak response to glucose was mea-

sured from the insulin concentration in fraction 6. The second phaseresponse was calculated from the mean insulin content in fractions10–14 while maintaining the high glucose concentration. The cells werefinally stimulated with a mixture of 16.7 mmol/L glucose and 10mmol/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 orsix perifusion lines were run in parallel using a multichannel perifusionapparatus (Brandel, Gaithersburg, MD).

Statistical methods

Differences between groups were tested with StatView 4.1 softwarefor the Macintosh (Abacus Concepts, Berkeley, CA), using one-wayANOVA 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 prototypestrains of CBV-3, CBV-4/J.V.B., CBV-5, and CAV-9 and thediabetogenic strain, CBV-4-E2. Virus replication was studiedby measuring the infectivity of samples collected at differentintervals. The results showed that all viruses replicated wellin all human islet preparations tested (Fig. 1).

In all CBV-infected cultures the first morphologicalchanges were seen by 1–2 days after infection, whereas CAV-9-infected cells seemed to be virtually intact even at 7 daysafter infection. Cells infected with CBVs first became morerounded and then they detached from the islet into the me-dium. As a result, the size of the islets decreased graduallyduring infection. After longer incubation, the virus-inducedcytopathic effect became more pronounced, but some isletcells remained intact over the entire observation period. In-fection-induced damage was also evident by the live-deadcell assay. At 4.5 days after infection the whole islet wascovered by dead cells, as shown in Fig. 2 for the prototypestrain of CBV-4/J.V.B.

FIG. 1. Replication of enteroviruses inadult human islets. Parallel aliquots offree floating islets were infected withapparent high multiplicity (30–100plaque-forming units/well) of CBV-4-E2, CBV-3, CBV-4/J.V.B., CBV-5, andCAV-9. After a 1-h adsorption periodthe inoculum virus was removed, isletswere washed twice, and culture me-dium was added. Samples taken at dif-ferent intervals were assayed for totalinfectivity. The results are shown as themean 6 95% confidence intervals ofthree experiments.

434 ROIVAINEN ET AL. JCE & M • 2000Vol 85 • No 1

To confirm that replication of enteroviruses took place ininsulin-producing b-cells, we used a double antibody im-munofluorescence technique with insulin and virus-specificantisera. Binding of antibodies was visualized by FITC- andLRSC-labeled conjugates. Virus-infected insulin-containingcells were readily identified by their yellow or orange color(Fig. 3).

Electron microscopy of human islets revealed that after 2days of infection, about 40% of CBV-5 infected cells hadundergone morphological changes characteristic of pykno-sis, i.e. highly distorted and wrinkled nuclei that often weredisplaced to the cellular periphery. These nuclei had a cres-cent shape, with deep indentations and highly condensedchromatin (Fig. 4A). There were no signs of nuclear or cy-toplasmic fragmentation or of crescent-shaped chromatincondensations, both known to be characteristic of apoptosis.Plasma membranes were intact in these cells. A similar mor-phology was not found in control cells. One week after in-fection, the majority of cells showed characteristics of sec-ondary necrosis. In the pyknotic cells, polyhedral virusparticles measuring approximately 30 nm could be observedin the cytoplasm (Fig. 4B).

Insulin release and content

As expected, uninfected adult human b-cells responded toglucose with a biphasic response of insulin release, with the

first phase peak (4.1 6 0.9-fold over the basal level) occurringin the first poststimulatory fraction, followed by a prolongedsecond phase (2.6 6 0.5-fold over the basal level). Finally, theglucose response was further potentiated by 10 mmol/Ltheophyline (6.9 6 1.2-fold).

CBV infection induced a readily detectable perturbance ininsulin release. The results of experiments performed 7 daysafter infection are summarized in Fig. 5. The most deleteriousviruses were CBV-3 and CBV-5. In all experiments the glu-cose responses of islets infected with these viruses weresignificantly decreased within 1 week (Fig. 5A). The suscep-tibility of the cells to CBV-4 strains was somewhat morevariable. As a result, only CBV-3 and CBV-5 significantlyaffected the first and second phase responses to glucose. Bothstrains of CBV4 also impaired the response to theophylineplus glucose. Unlike CBV-infected islets, CAV-9-infectedcells responded well to both stimuli at 1 week after infection(Fig. 5A).

Insulin content was studied from the aliquots of samplesharvested at different time points for perifusion. Resultsobtained on day 7 are summarized in Fig. 5B. At this time,the insulin content per cellular DNA of islets infected byCBV4-E2 and CBV-5 had decreased by about 40%, and thatof those infected by CBV-3 had decreased by 65% comparedwith that in uninfected control islets. The effect of CBV-4/J.V.B. did not reach statistical significance. Analogous with

FIG. 2. Effect of enterovirus infectionon human islet cell viability. CBV-4/J.V.B.-infected and uninfected controlislets were harvested at 2 and 4.5 daysafter infection and stained with the live/dead cell assay kit. Live cells arestained green by calcein due to their es-terase activity, whereas red fluores-cence is induced in the nuclei of deadcells by ethidium homodimer-1. Cofluo-rescence from adjacent overlappingcells results in a yellow color. Peripheralislet cells are dying at 4.5 days of infec-tion. The figure is representative of ninesimilar experiments.


the insulin release data, the insulin content of CAV-9-in-fected cells remained intact.

DNA fragmentation

Based on TUNEL, only a minority of the infected cellsbecame apoptotic (Fig. 6). However, at 2 days after infection,DNA fragmentation in the nuclei of b-cells was significantlyincreased in CBV-5-infected cells (5.9 6 1.1%; P , 0.001) andtended to be increased also in CBV-4-infected cells (3.9 60.5%; 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 ofTUNEL-positive cells after 7 days, when the majority ofCBV-infected cells had died or had pyknotic nuclei as de-termined by electron microscopy.

iNOS mRNA expression

The possibility that NO mediates infection-induced b-cellimpairment was studied by measuring iNOS mRNA expres-sion and nitrite accumulation in the culture medium. Thecellular content of iNOS mRNA was determined by semi-quantitative RT-PCR of mRNA isolated from CBV-5-infectedcells and controls. As a positive control, the islets were stim-ulated for 24 h with interferon-g (1000 U/mL) and interleu-kin-1b (30 U/mL). One day of CBV-5 infection did not mod-ify iNOS expression, whereas culture in the presence of thecytokines induced a 5-fold increase in human islet iNOSexpression (Fig. 7). A 2-day viral infection also failed to increaseiNOS mRNA expression (data not shown), and there was noconsistent increase in medium nitrite accumulation after 2–10days of infection with CBV-5 or CBV-3 (data not shown).

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. Harvestedcells were cytocentrifuged on glass slides, fixed with cold methanol, and double stained with enterovirus-specific rabbit antiserum andinsulin-specific sheep antiserum. Visualization was performed by FITC-antirabbit (green, for virus antigens) and LRSC- antisheep (red, forinsulin) conjugates. Yellow-orange double fluorescence is seen after infection with all viruses, indicating that the viruses have effectively infectedthe b-cells. A and B are representative of nine and three similar experiments, respectively. CBV-3-infected islets together with uninfectedcontrols were studied in separate experiments and analyzed by confocal microscopy.


Discussion

The present study shows that in addition to the diabeto-genic strain E2 of CBV-4, the prototype strains of CBV-3,CBV-4, CBV-5, and CAV-9 are able to infect insulin-produc-ing b-cells in primary adult human islets. The insulin releaseresponses to secretagogues were markedly impaired in CBV-infected b-cells, even when their insulin content was onlymarginally decreased. These effects were partly due to b-cell

lysis, but the remaining b-cells also appeared to be func-tionally impaired. CBV-3 and CBV-5 induce the most severesignals of b-cell dysfunction and damage, whereas CAV-9infection did not cause any apparent adverse effects on b-cellfunction during the 7-day follow-up.

Enteroviruses are classically associated with lytic infec-tions, but they can also establish noncytolytic or chronicinfections (22, 35). Recently two types of death mechanismswere reported for one enterovirus, poliovirus type 1 (36). Itwas shown that productive virus infection in HeLa cellsresults in pyknosis characterized by highly distorted nucleiwith condensed, but intact, chromatin, whereas conditionsrestricting viral production are associated with typical apo-ptosis. Our ultrastructural observations demonstrated thatCBV-5-infected cells died by the process of pyknosis and notby apoptosis. Only a minority of the infected cells becameapoptotic, as evidenced by nuclear morphology and in-creased in situ DNA end labeling. This suggests that apo-ptosis may not have a major role in CBV-5-induced cell deathduring a productive infection.

Nitric oxide may be a mediator of b-cell death in diabetesmellitus (reviewed in Ref. 37), and there is evidence that viralinfection leads to NO production by different cell types (38,39). In the present experiments there was no direct inductionof iNOS expression and nitrite production by CBV-5 infec-tion, suggesting that virally induced b-cell death was notmediated by NO. It cannot, however, be excluded that viralinfection in vivo, accompanied by local production of cyto-kines by infiltrating immune cells, will lead to islet NOproduction.

Unlike CBVs, CAV-9 appeared to cause a noncytolyticinfection. It replicated well in human b-cells, and the infectedislets still responded like uninfected control cells at 1 weekafter infection. The effects of CAV-9 were studied because itis genetically closely related to the CBVs (40), but its bio-logical effects are distinct. In newborn mice, for example, itaffects only skeletal muscle, whereas CBVs are capable ofaffecting several other organs as well (41). Most importantly,CAV-9 was one of the enteroviruses found to be temporallyassociated with increases in the levels of islet cell antibodiesin prediabetic children (42). Based on our findings, per-sistent CAV-9 infection of the b-cells could be one mech-anism linking enterovirus infections with b-cell targetedautoimmunity.

Some heterogeneity was apparent in the susceptibility ofthe b-cells to the effects of enteroviruses even within a singleexperiment. Although some b-cells were lysed, neighboringb-cells remained virtually intact in the CBV-infected cul-tures. This is in accordance with the well known situation atthe onset of diabetes, when some islets may still be intactwhile in others only noninsulin-producing cells remain (re-viewed in Ref. 43). Similar observations have also been re-ported previously with infection of cultured human isletswith the diabetogenic strain E2 of CBV-4 (1). This couldreflect the metabolic heterogeneity of the b-cells. Thus, onlya proportion of b-cells becomes metabolically activated dur-ing glucose stimulation (44), and this active b-cell subpopu-lation is preferentially inhibited by the cytokine interleu-

FIG. 4. Electron microscopy of infected human islets. A, Electronmicrograph of CBV-5-infected cells (2 days; magnification, 34000)showing a b-cell (left) and an a-cell (right). Both cells show morpho-logical evidence of pyknosis, i.e. distorted nucleus with condensedchromatin. B, Higher magnification (340 000) of a CBV-5-infected cellshowing virus particles in the cytoplasm. The arrowheads point tovirus particles.


kin-1b (45). Whether the metabolically active b-cells are alsothe ones most severely affected by enteroviruses remainsunknown at present.

The induction of virus-induced diabetes in mice is knownto depend on the genetic background of the host and thepassage history of the virus (46, 47). The human isolate E2 ofCBV-4 can induce a diabetes-like syndrome in mice. The

prototype strain of CBV-4/J.V.B. is able to replicate in murinepancreatic b-cells, but it does not cause cell lysis or produceglucose abnormalities (17, 47). However, diabetogenicity ofthis virus strain can be enhanced by passaging it either in vivoin mouse pancreas or in b-cell cultures (16, 17). In contrast tomurine pancreatic b-cells, human adult b-cells were foundhere to be susceptible to the cytolytic effects of the prototype

FIG. 5. A, Stimulated release of insulinin adult human islet cells in perifusionexperiments at 7 days after enterovirusinfection. Results are shown for the cu-mulative data obtained with nine hu-man islet preparations. Insulin releaseis expressed as the stimulation index(stimulated/basal level) in response to16.7 mmol/L glucose, first (GLUG 1)and second (GLUG 2) phases separately(see Materials and Methods for theirdefinition), and to glucose plus 10mmol/L theophyline (G1T). B, Insulincontent per cellular DNA of the humanislets harvested at 7 days after infec-tion, expressed as relative changes fromthe uninfected control. The number ofobservations is indicated at the bottomof the columns. *, P , 0.05; **, P , 0.01(compared with the uninfected controlcells).


strains of CBV-4/J.V.B. and CBV-5. The reasons for the ob-served differences between species are not known at present.

The most deleterious viruses in adult human islets wereCBV-3 and CBV-5. CBV-5 is known to occur as explosiveepidemics (11). Interestingly, increased incidence of IDDMhas been reported after epidemics of CBV-5 (10, 11), and

CBV-5 epidemics occur frequently in Finland, where theincidence of IDDM is the highest in the world (48).

Although some potentially diabetogenic strains of entero-viruses have been described (1, 15), there is no strong evi-dence to suggest that the putative diabetogenic propertywould be restricted to a single or even a few strains orserotypes only. It is possible that when infecting a geneticallysusceptible individual, several different serotypes or perhapseven all enteroviruses could be diabetogenic. The large num-ber of different enterovirus serotypes makes identification ofthe most pathogenic serotypes an important, but demanding,task. The screening process could be simplified significantlyby standardized experiments with primary islet cultures, aspresently presented.

In conclusion, we have shown that several prototypestrains of enteroviruses infect human b-cells. The responsesof infection were different from those previously reported inrodent b-cells. In human b-cells, CBV typically cause a lyticinfection, characterized by nuclear pyknosis, but only someof the b-cells are immediately killed. The functional capacityof the remaining b-cells is also deteriorated. CoxsackievirusA9 represents a noncytolytic type of infection in the b-cell.Such an infection could theoretically lead to the initiation orexaggeration of b-cell-targeted autoimmunity.

Acknowledgments

We thank Prof. J.-W. Yoon for the diabetogenic E2 strain of CBV-4used in the study, Ms. Mervi Eskelinen for skillful technical assistance,and the personnel of the b-Cell Transplant (Brussels, Belgium) for thepreparation of human islet cells.

FIG. 6. TUNEL staining for the simultaneous in situ detection of DNA fragmentation (black nuclear staining), and insulin (red-browncytoplasmic 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 withcontrol.

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 withCBV-5. The results of densitometric quantitation of iNOS/GAPDHexpression are shown at the bottom. The figure is representative ofthree similar experiments.


References

1. Yoon J-W, Austin M, Onodera T, Notkins AL. 1979 Isolation of a virus fromthe pancreas of a child with diabetic ketoacidosis. N Engl J Med. 300:1173–1179.

2. Yoon J-W. 1990 The role of viruses and environmental factors in the inductionof diabetes. Curr Top Microbiol Immunol. 164:95–123.

3. King ML, Shaikh A, Bidwell D, Voller A, Banatvala JE. 1983 Coxsackie-B-virus-specific IgM responses in children with insulin-dependent (juvenile-onset; type 1) diabetes mellitus. Lancet. 1:1397–1399.

4. Banatvala JE, Bryant J, Schernthaner G, et al. 1985 Coxsackie B, mumps,rubella and cytomegalovirus specific IgM responses in patients with juvenile-onset insulin-dependent diabetes mellitus in Britain, Austria, and Australia.Lancet. 1:1409–1412.

5. Frisk G, Nilsson E, Tuvemo T, Friman G, Diderholm H. 1992 The possiblerole of Coxsackie A and Echo viruses in the pathogenesis of type 1 diabetesmellitus studied by IgM analysis. J Infect Dis. 24:13–22.

6. Helfand RF, Gary Jr HE, Freeman CY, Anderson LJ, Pittsburgh DiabetesResearch Group, Pallansch MA. 1995 Serological evidence of an associationbetween enteroviruses and the onset of type 1 diabetes mellitus. J Infect Dis.172:1206–1211.

7. Dahlquist G, Ivarsson S, Lindberg B, Forsgren M. 1995 Maternal enteroviralinfection during pregnancy as a risk factor for childhood IDDM. A population-based case-control study. Diabetes. 44:408–413.

8. Hyoty H, Hiltunen M, Knip M, et al. 1995 A prospective study on the role ofcoxsackie B and other enterovirus infections in the pathogenesis of IDDM.Diabetes. 44:652–657.

9. Hiltunen M, Hyoty M, Knip M, et al. 1997 ICA seroconversion in children istemporally associated with enterovirus infections. J Infect Dis. 175:554–560.

10. Rewers M, LaPorte RE, Walczak M, Dmochowski K, Bogaczynska E. 1987Apparent epidemic of insulin-dependent diabetes mellitus in midwesternPoland. Diabetes. 36:106–113.

11. Wagenknecht LE, Roseman JM, Herman WH. 1991 Increased incidence ofinsulin-dependent diabetes mellitus following an epidemic of coxsackievirusB5. Am J Epidemiol. 133:1024–1031.

12. Cherry JD. 1987 Enteroviruses: polioviruses (poliomyelitis), coxsackieviruses,echoviruses, and enteroviruses. In: Feigin RD, Cherry JD, eds. Textbook ofpediatric infections. Philadelphia: Saunders; 1729–1790.

13. Jenson A, Rosenberg B, Notkins AL. 1980 Pancreatic islet-cell damage inchildren with fatal viral infections. Lancet. 2:354–358.

14. Ujevich MM, Jaffe R. 1980 Pancreatic islet cell damage. Its occurrence inneonatal coxsackievirus encephalomyocarditis. Arch Pathol Lab Med.104:438–441.

15. Champsaur H, Dussaix E, Samolyk D, Fabre M, Bach CH, Assan R. 1980Diabetes and coxsackie virus B5 infection. Lancet. 2:251.

16. Toniolo A, Onodera T, Jordan G, Yoon J-W, Notkins AL. 1982 Virus-induceddiabetes mellitus. Glucose abnormalities produced in mice by the six membersof the coxsackie B virus group. Diabetes. 31:496–499.

17. Szopa TM, Gamble DR, Taylor KW. 1985 Biochemical changes induced bycoxsackie B4 virus in short-term culture of mouse pancreatic islets. Biosci Rep5:63–69.

18. Yoon J-W, London WT, Curfman BL, Brown RL, Notkins AL. 1986 Coxsackievirus B4 produces transient diabetes in nonhuman primates. Diabetes.35:712–716.

19. Yoon J-W, Onodera T, Jenson AB, Notkins AL. 1978 Virus-induced diabetesmellitus. XI. Replication of coxsackie B3 virus in human pancreatic b cellcultures. Diabetes. 27:778–781.

20. Vuorinen T, Nikolakaros G, Simell O, Hyypia T, Vainionpaa R. 1992 Cox-sackievirus B3 and mumps virus infection in human fetal islet cell cultures.Pancreas. 7:460–464.

21. Gerling I, Chatterjee NK. 1990 Autoantigen (64 000-Mr) expression in cox-sackievirus B4-induced experimental diabetes. Curr Top Microbiol Immunol.156:55–62.

22. See DM, Tilles JG. 1995 Pathogenesis of virus-induced diabetes in mice.J Infect Dis. 171:1131–1138.

23. Kaufman DL, Erlander MG, Clare-Salzler M, Atkinson MA, Maclaren NK,Tobin AJ. 1992 Autoimmunity to two forms of glutamate decarboxylase ininsulin-dependent diabetes mellitus. J Clin Invest. 89:283–292.

24. Lonnrot M, Hyoty H, Knip M, et al. 1996 Antibody cross-reactivity inducedby the homologous regions in glutamic acid decarboxylase (GAD65) and 2Cprotein of coxsackievirus B4. Clin Exp Immunol. 104:398–405.

25. Eizirik DL, Pipeleers DG, Ling Z, et al. 1994 Major species differences betweenhumans and rodents in the susceptibility to pancreatic b-cell injury. Proc NatlAcad Sci USA. 91:9253–9256.

26. Welsh N, Margulis B, Borg LAH, et al. 1995 Differences in the expression ofheat-shock proteins and antioxidant enzymes between human and rodentpancreatic islets: implications for the pathogenesis of insulin-dependent dia-betes mellitus. Mol Med. 1:806–820.

27. Keymeulen B, Ling Z, Gorus FK, et al. 1998 Implantation of standardizedb-cell grafts in a liver segment of IDDM patients: graft and recipient charac-teristics in two cases of insulin-independence under maintenance immuno-suppression for prior kidney graft. Diabetologia. 41:452–459.

28. Lennette EH. 1969 General principles underlying laboratory diagnosis of viraland rickettsial infections. In: Lennette EH, Schmidt NJ, eds. Diagnostic pro-cedures for viral and rickettsial infections. New York: American Public HealthAssociation; 1–63.

29. Hovi T, Roivainen M. 1993 Peptide antisera targeted to a conserved sequencein poliovirus capsid protein VP1 cross-react widely with members of the genusEnterovirus. J Clin Microbiol. 31:1083–1087.

30. Hinegardner RT. 1971 An improved fluorometric assay for DNA. Anal Bio-chem. 39:197–201.

31. Otonkoski T, Beattie GM, Mally MI, Ricordi C, Hayek A. 1993 Nicotinamideis a potent inducer of endocrine differentiation in cultured human fetal pan-creatic cells. J Clin Invest. 92:1459–1466.

32. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, TannenbaumSR. 1982 Analysis of nitrate, and [15N]nitrate in biological fluids. Anal Bio-chem. 126:131–138.

33. Pavlovic D, Chen M-C, Bouwens L, Eizirik DL, Pipeleers D. 1999 Contri-bution of ductal cells to cytokine responses by human pancreatic islets. Dia-betes. 48:29–33.

34. Otonkoski T, Hayek A. 1995 Constitution of a biphasic insulin response toglucose in human fetal pancreatic b-cells with glucagon-like peptide 1. J ClinEndocrinol Metab. 80:3779–3783.

35. Vella C, Brown CL, McCarthy DA. 1992 Coxsackievirus B4 infection of themouse pancreas: acute and persistent infection. J Gen Virol. 73:1387–1394.

36. Agol VI, Belov GA, Bienz K, et al. 1998 Two types of death of poliovirus-infected cells: caspase involvement in the apoptosis but not cytopathic effect.Virology. 252:343–353.

37. Eizirik DL, Pavlovic D. 1997 Is there a role for nitric oxide in b-cell dysfunctionand damage in IDDM? Diabetes Metab Rev. 13:293–307.

38. Nathan CF. 1995 Natural resistance and nitric oxide. Cell. 82:873–876.39. Heitmeier MR, Scarim AL, Corbett JA. 1998 Double-stranded RNA-induced

inducible nitric oxide synthase expression and interleukin-1 release by murinemacrophages requires NF-kB activation. J Biol Chem. 273:15301–15307.

40. Chang KH, Day C, Walker J, Hyypia T, Stanway G. 1992 The nucleotidesequence of wild-type coxsackievirus A9 strains imply that RGD motif in VP1is functionally significant. J Gen Virol. 73:621–626.

41. Hyypia T, Kallajoki M, Maaronen M, et al. 1993 Pathogenic differencesbetween coxsackie A and B virus infections in newborn mice. Virus Res.27:71–78.

42. Roivainen M, Knip M, Hyoty H, et al. 1998 Several different enterovirusserotypes can be associated with prediabetic autoimmune episodes and onsetof overt IDDM. J Med Virol. 56:74–78.

43. Pipeleers D, Ling Z. 1992 Pancreatic b-cells in insulin-dependent diabetes.Diabetes Metab Rev. 8:209–227.

44. Pipeleers DG. 1992 Heterogeneity in pancreatic b-cell population. Diabetes.41:777–781.

45. Ling Z, Chen M-C, Smismans A, et al. 1998 Intercellular differences in in-terleukin-1b-induced suppression of insulin synthesis and stimulation of non-insulin protein synthesis by rat pancreatic b-cells. Endocrinology.139:1540–1545.

46. Notkins AL, Yoon J, Onodera T, Jenson AB. 1979 Virus-induced diabetesmellitus: infection of mice with variants of encephalomyocarditis virus, cox-sackievirus B4, and reovirus type 3. Adv Exp Med Biol. 119:137–147.

47. Yoon J-W, Onodera T, Notkins AL. 1978 Virus-induced diabetes mellitus. XV.b Cell damage and insulin-dependent hyperglycemia in mice infected withcoxsackie B4. J Exp Med. 148:1068–1080.

48. Hovi T, Stenvik M, Rosenlew M. 1996 Relative abundance of enterovirusserotypes in sewage differs from that in patients: clinical and epidemiologicalimplications. Epidemiol Infect. 116:91–97.


Mechanisms of Coxsackievirus-Induced Damage to Human Pancreaticβ -Cells 1

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