Adenoviral-Mediated Transduction of Human Pancreatic Islets: Importance of Adenoviral Genome for Cell Viability and Association with a Defic
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内分泌学杂志 2005年第5期
Departments of Medical Cell Biology (A.R.B., N.W.) and Medical Biochemistry and Microbiology (G.A.), Uppsala University, S-751 23 Uppsala, Sweden
Address all correspondence and requests for reprints to: Dr. Andreea Barbu, Department of Medical Cell Biology, Biomedical Center, P.O. Box 571, S-751 23 Uppsala, Sweden. E-mail: andreea.barbu@medcellbiol.uu.se.
Abstract
As adenoviral vectors are extensively used for genetic manipulation of insulin-producing cells in vitro, there is an increasing need to evaluate their effects on the function, morphology, and viability of transduced pancreatic islets. In the present study we observed that specific adenoviral genotypes, carrying E4 and E1/E3 deletions, correlate with differential induction of necrosis in pancreatic islet cells. In particular, the adenovirus death protein encoded from the E3 region of the adenoviral genome was able to modulate the changes induced in the morphology and viability of the transduced cells. We also propose a putative role for the transcriptional regulator pIX. Although human islet cells showed an increased resistance in terms of viral concentrations required for the induction of cell toxicity, our results showed that they were unable to build up an efficient antiviral response after transduction and that their survival was dependent on the exogenous addition of -interferon. An intact and fully functional ?-cell is crucial for the successful application of gene therapy approaches in type 1 diabetes, and therefore, the implications of our findings need to be considered when designing vectors for gene transfer into pancreatic ?-cells.
Introduction
TYPE 1 DIABETES IS an autoimmune disease that eventually causes complete destruction of insulin-secreting ?-cells in the pancreas. Recently, pancreatic islet transplantation has been validated as a promising alternative to correct for the insulin deficiency in type 1 diabetes (1). However, the requirement for islets derived from two to four donors to treat a single patient is a barrier to the use of this technique on a larger scale. As a result, much effort is going into developing new therapeutic strategies to increase durable functional islet mass. Gene therapy offers a potential means to protect against ?-cell death by induction of immunoregulatory, cytoprotective, or antiapoptotic genes (2, 3) as well as to stimulate ?-cell differentiation and regeneration by induction of different transcriptional regulators and growth factors (4, 5, 6).
The ability to engineer pancreatic ?-cells is a prerequisite for successful application of gene therapy approaches in type 1 diabetes. However, pancreatic islets are terminally differentiated cell clusters that are difficult to reach in vivo and troublesome to maintain in tissue culture. Therefore, the use of nonviral transfection strategies for genetic modification of insulin-producing cells, including lipofection, electroporation, and biolistic particles, mediate only low transfection efficiencies (7, 8). In contrast, due to their capacity to mediate highly efficient gene transfer in nondividing cells, viral vectors have emerged as the first choice for engineering ?-cells. Together with lentiviruses (9) and adeno-associated viruses (10), adenoviruses are the most commonly used viral vectors in current ?-cell research (11, 12). However, despite their advantages, such as high titer production and the diminished risk for insertional mutagenesis due to the epichromosomal location within the target cell, adenoviral transduction and gene transfer may interfere with ?-cell function and/or induce cell death. Adenoviruses contain genes that are able to initiate and modulate cell death by inducing or suppressing specific processes within the target cell, and these effects are likely to be dependent on both the genotype of the viral vector used and the cell system studied (13). Adenoviral genes are transcribed in a complex temporal manner. Early genes are transcribed from five different promoters, in the order E1A, E4, E3, E1B, and E2. These genes are mainly concerned with the regulation of viral gene expression and DNA replication and the modulation of cell death. The E3–11.6K protein, also known as the adenovirus death protein (ADP), is encoded by the E3 transcription unit of the adenoviral genome, but its expression is turned on preferentially by a strong adenovirus major late promoter. In contrast to the other antiapoptotic genes located in the same region, ADP has been shown to facilitate viral exit from the nucleus of the infected cells by inducing cell death (14). The early production of interferons (IFNs) is believed to be an important host response to many viral infections, including adenoviruses (15), and, in pancreatic islets, IFN production has been associated with hyperexpression of major histocompatibility complex (MHC) class IA antigens (16, 17).
Previously, we reported that E1/E3-deleted adenoviral vectors induce ?-cell cytotoxicity (12). In the present study we investigate the effects of various adenoviral genome deletions on rat and human islet cell viability to determine whether a specific adenoviral genotype correlates with ?-cell death. We also try to determine whether islet cells are able to establish an antiviral state in response to adenoviral transduction and the role of type I IFNs during this process.
Materials and Methods
Reagents
RPMI 1640, fetal calf serum (FCS), L-glutamine, penicillin/streptomycin, trypsin-EDTA, bisbenzimide (Hoechst-33342), and propidium iodide were purchased from Sigma-Aldrich Corp. (St. Louis, MO). All other reagents used, unless otherwise specified, were obtained from E. Merck (Darmstadt, Germany).
Cell culture
Rat pancreatic islets of Langerhans were isolated from 3-month-old Sprague Dawley rats (local Uppsala colony) by a collagenase digestion procedure and cultured in groups of 150/50-mm well in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, benzylpenicillin (100 U/ml), and streptomycin (0.1 mg/ml), containing 11.1 mM glucose (18). Human pancreatic islets, provided by Prof. Olle Korsgren (Uppsala University, Uppsala, Sweden), were cultured in the medium described above, containing 5.6 mM glucose, at 37 C in humidified air with 5% CO2. Culture medium was replaced every 48 h. The use of rat and human pancreatic islets was in accordance with international guidelines (NIH publication 85-23, revised 1985) and was approved by the local ethics committees for Uppsala University.
Adenoviral vectors and viruses
The following human adenovirus (Ad) serotype 5 mutants were used in this study: 1) AdCMVProg, from a progesterone-inducible system, in which the cytomegalovirus (CMV) promoter drives the cDNA for an inactive transactivator protein (genotype E1/E3-deleted 455–3328 and 30005–30750) (19); 2) Adeno-X-Tet-Off (BD Clontech Laboratories, Inc., Palo Alto, CA), encoding a tetracycline-controlled transactivator protein (genotype E1/E3-deleted, 342–3528 and 27865–30995, with bigger deletions both in the E1 region, encompassing a portion of the pIX promotor, and in the E3 region of the adenoviral genome, encompassing ADP) (20); and 3) dl366, E4-deleted, expressing none of the products of the early region 4 (21). The Adeno-X-Tet-Off viral vector is similar to the AdEasy-1 vector, but not identical, because AdEasy-1 is deleted in nucleotides 480-3533 and 28130–30820 (22). All virus stocks were purified by cesium chloride density gradient centrifugation (L-80 ultracentrifuge, Beckman Coulter, Fullerton, CA) and plaque-titrated by serial dilution and agar overlay on their respective transcomplementing cell lines. Typical titers were 109 plaque-forming units (PFU)/ml or higher, representing 1–5% of the total viral particles as determined by readings of the ODs.
The E3-ADP variant adenoviruses used in this study, rec700 and pm734.1, were provided by Prof. William S. M. Wold (St. Louis University School of Medicine, St. Louis, MO). rec700 is an Ad5-Ad2-Ad5 recombinant (23) and is the parental virus for pm734.1 (with ADP residues 1–48 deleted) (14).
Adenoviral transduction, viral transduction, and assessment of transfection efficiency
Rat and human islets were dispersed into individual cells by treatment with trypsin (5 mg/ml) for 5 min, in Ca2+- and Mg2+-free Hanks’ solution and were cultured for an additional 1 h at 37 C. Single cells from dispersed islets were transduced with adenoviral vectors or wild-type viruses in a minimum volume of 0.1 ml RPMI 1640 supplemented with 2% FCS, containing various adenoviral concentrations (PFU per cell). After 1-h incubation at 37 C, islet cells were washed with RPMI 1640 medium and plated in 96- or 24-well plates, coated with 0.1% gelatin. The plates were incubated at 37 C, and medium was changed every second day for up to 1 wk post transduction. In some cases, 24 h before transduction and during the experiments, islet cells were treated with 1000 U/ml rat or human IFN- (PBL Biomedical Laboratories, Piscataway, NJ).
To assess the efficiency of the adenoviral-induced ?-cell transduction, we coincubated intact islets and dispersed rat islet cells with 100 PFU/cell of an adenoviral vector expressing the ?-galactosidase gene under control of the CMV promoter. The next day islets were dispersed, cells were fixed, and enzymatic activity and transduction efficiency were determined using light microscopy studies as previously described (24). To evaluate the capacity of wild-type adenoviruses to infect dispersed islet cells, we performed fluorescence microscopy studies after staining for the E2–72kD adenovirus DNA binding protein (25).
Sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT) assay
Adenoviral-induced cell death was assessed using the colorimetric assay (XTT-based) for cell viability (12). Adenoviral-transduced and nontransduced rat and human islet cells were plated in 96-well plates in 100 μl complete RPMI 1640 culture medium. Three days after transduction when using E3-ADP variant adenoviruses or 7 d after transduction when using recombinant adenoviral vectors, 50 μl 1 mg/ml XTT (Roche, Mannheim, Germany) was added to each well. After 5 h of incubation at 37 C, the ODs were read in a Microplate autoreader (Bio-Tek Instruments, Inc., Watford, UK) at 490 nm.
Fluorescence microscopy
To investigate the effect of adenoviruses on islet cell viability, we performed fluorescence microscopy studies after 3–7 d (rat islet cells) and 5–7 d (human islet cells) of culture post transduction. Rat and human islet cells were incubated in RPMI 1640 complete medium containing 5 μg/ml bisbenzimide and 10 μg/ml propidium iodide for 10 min at 37 C. The cells were then washed, detached by mild trypsination, and examined by fluorescence microscopy.
RNA extraction and cDNA synthesis
Twenty to 24 h after transduction of rat and human islets with adenoviruses, total cellular RNA was isolated by a single-step method, using a commercial system (Ultraspec RNA isolation system, Biotecx Laboratories, Inc., Houston, TX), followed by digestion with ribonuclease-free deoxyribonuclease (Qiagen, Chatsford, CA). The total RNA for each sample was reverse transcribed with Moloney murine leukemia virus reverse transcriptase using an oligo(deoxythymidine) primer in the presence of 5 mM MgCl2, 1 mM of each deoxy-NTP, and 1 U/μl recombinant RNasin ribonuclease inhibitor. The RT reaction was performed at 42 C for 40 min, followed by inactivation of the enzyme at 95 C for 5 min.
Real-time PCR analysis
On the basis of the respective rat and human cDNAs, primers were designed to amplify specific regions of Ifn-, class IA MHC antigen-processing and antigen presentation genes [?2-microglobulin (?2m), low molecular mass polypeptide 2 (Lmp-2), and transporter associated with antigen processing 1 (Tap-1) for human islet cells; ?2m, Lmp-7, and Tap-1 for rat islet cells], and the ?-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs. PCR amplifications were carried out in a real time LightCycler (Roche) using the temperature-time sequence: denaturation for 15 sec at 95 C, annealing for 13 sec at 55 C, and extension for 6 sec at 72 C, and the primer sequences described in Table 1.
TABLE 1. PCR primers used to assess gene expression in human and rat islet cells
To obtain relative mRNA expression values, PCRs were performed with ?-actin for rat samples and GAPDH or ?-actin for human samples for each of the experimental groups. The amount of PCR product relative to these housekeeping genes was calculated as previously described (26). For each experiment the data are presented as normalized values against the relative mRNA levels of the control samples. All PCR products were verified by agarose gel electrophoresis and SYBR Gold (Molecular Probes, Eugene, OR) staining.
Statistical analysis
Data are summarized as the mean ± SEM. The significances of differences between the groups were determined by one- or two-way ANOVA for repeated measurements and the Bonferroni or Dunnett’s test. Differences were considered significant at P < 0.05. Statistical analysis was performed using SigmaStat (SPSS Science Software, Erkrath, Germany).
Results
To evaluate the efficacy of adenoviral vectors to transduce islet cells, we stained for ?-galactosidase activity and counted cells in a light microscope. In a typical experiment we observed more than 90% ?-galactosidase-positive cells in the dispersed islet cell group and only 20–30% ?-galactosidase-positive cells in the intact islet cell group (results not shown). In all subsequent transduction experiments, only dispersed islet cells were used to ensure high transduction efficiency. The adenoviral vector AdCMVProg belongs to an inducible system that we have previously described (12). We showed that increasing concentrations of the virus resulted in a corresponding increase in protein expression levels, with more than 70% ?-cells transduced at 100 PFU/cell. We obtained similar transduction efficiencies (>80%) with another adenoviral vector expressing green fluorescent protein that has the same backbone as the AdTetOFF vector used in the present study.
Effects of different adenoviral vector genomes on rat and human islet cell viability
To determine whether the adenoviral-induced cytotoxicity correlates to a particular adenoviral genotype, we transduced dispersed rat and human islet cells with different replication deficient adenoviral vectors carrying deletions in one or more of the E1, E3, or E4 regions of the adenoviral genome.
XTT-based cell viability assays performed 7 d after adenoviral vector/islet cell incubation, revealed that transduction of rat islet cells with dl366 virus (carrying intact E1 and E3 regions, but none of the genes of the E4 region) significantly diminished islet cell viability (to 61.8 ± 0.038%) at 1 PFU/cell and that the adenoviral-induced cell death occurred in a dose-dependent manner (with no viable cells left at 100 PFU/cell; Fig. 1A,
). To investigate whether this cytotoxicity was due to nonspecific effects of proteins belonging to the adenoviral capsids we coincubated islet cells with heat-inactivated dl366 viruses. One week post transduction, no effect on the cell viability was observed (results not shown), suggesting that adenovirus-induced cell death is secondary to the internalization of functional vector particles into the cells. Adenovirus-mediated toxicity was also seen after transduction of islet cells with the E1/E3-attenuated viral vector, AdCMVProg (Fig. 1B,
), although to a lesser extent. Thus, 1–10 PFU/cell of the E4 dl366 virus induced 50% cytotoxicity in rat islet cells, whereas in AdCMVProg-transduced cells the same effect was obtained only at concentrations as high as 10–100 PFU/cell. Interestingly, transduction of rat islet cells with AdTetOFF vector, another E1/E3-deleted viral vector that carries additional deletions in the E1 region (encompassing a portion of the pIX promotor) and in the E3 region (encompassing ADP), resulted in a lesser induction of islet cell death and only at a high concentration of the vector (Fig. 1C,
).
FIG. 1. Decreased islet cell viability in response to dl366 (A), AdCMVProg (B), and AdTetOFF (C) adenoviral transduction. Dispersed human () and rat () islet cells were transduced with various concentrations of the adenoviral vectors, and cellular viability was assessed 7 d post transduction using an XTT-based assay. Individual colorimetric readings are normalized to the value of nontransduced islet cells. Results are the mean ± SEM of eight (rat islet cells) or 10 (human islet cells) separate experiments. * and #, P < 0.05; ** and ##, P < 0.01; *** and ###, P < 0.001 [vs. nontransduced human (*) or rat (#) cells, by one-way ANOVA, Bonferroni test]. D, ADP and pIX mRNA expression in rat and human islet cells after adenoviral exposure was analyzed by RT-PCR as described in Materials and Methods. The reaction was stopped during the exponential phase (23 cycles for ADP and pIX and 20 cycles for ?-actin), and the products were analyzed by ethidium bromide staining and agarose gel electrophoresis. E, Schematic of the adenoviral genome and the specific deletions of the adenoviral vectors used in this study: E1–E4, early transcribed adenoviral genes; MLP, major late promotor; VA-RNA, adenoviral RNA.
In human islet cells, transduction with dl366 vector induced a similar pattern of cytotoxicity, but at considerably higher viral concentrations (100 PFU/cell reduced cellular viability to 55.3 ± 10.9% in human islet cells vs. 0% in rat islet cells; Fig 1A, ). At high concentrations (100 PFU/cell) both E1/E3-attenuated adenoviruses reduced islet cell viability to 89.4 ± 3.9% when cells were transduced with AdTetOFF (Fig. 1C, ) and 54 ± 13.3% when cells were transduced with AdCMVProg (Fig. 1B, ). These results are in line with our previous observation that an adenoviral vector induces cytotoxicity in pancreatic islet cells (12) and suggest that this effect is modulated by the adenoviral genome and the particular characteristics of the target cells.
Effects of ADP on wild-type adenovirus-induced islet cell toxicity
RT-PCR analysis performed on mRNA isolated from transduced islet cells demonstrated a differential expression of the adenoviral genes IX and ADP. As anticipated, we could not detect ADP and IX cDNA in control or AdTetOFF cells (Fig. 1D). In addition, expression of the ADP and IX genes was clearly stronger in cells transduced with dl366 than in those transduced with AdCMVProg (Fig. 1D). Taken together, these data (Fig. 1) raise the possibility that the cytotoxic effect of adenoviruses in islet cells may be in part correlated to ADP expression. Indeed, after transduction of rat islet cells with an ADP– virus (pm734.1), we observed a milder effect on islet cell viability, as assessed by the XTT-based metabolic assay (Fig. 2A). Although not statistically significant, there was also a trend for a milder effect of the ADP– virus in human islet cells (Fig. 2B).
FIG. 2. Estimation of cellular cytotoxicity induced in dispersed islet cells by adenoviruses carrying differential expression of ADP. Rat islet cells (A) and human islet cells (B) were transduced with various concentrations of rec700 or pm734.1 virus. Cell viability was assessed on d 3 (rat islet cells) or d 5 post transduction (human islet cells) using the XTT-based assay. Individual colorimetric readings are normalized to the value of nontransduced islet cells. Results are the mean ± SEM of six separate experiments. Statistical significance was determined by two-way ANOVA and Dunnett’s test.
Fluorescence microscopic analysis after double staining with bisbenzimide/propidium iodide indicated that viral exposure promoted necrosis in both human (Fig. 3A) and rat (Fig. 3B) islet cells. The nuclei of the transduced cells were extremely distended, and this specific morphology preceded the rupture of the plasma membrane. Note that no increase in typical apoptotic features was observed in the transduced cells.
FIG. 3. Micrographs showing bisbenzimide/propidium iodide fluorescence of human (A) and rat (B) islet cells. Cells were transduced with 30 PFU/cell (rat cells) or 50 PFU/cell (human cells) of rec700 (wild type) and pm734.1 (ADP–), and cell morphology was evaluated by fluorescence microscopy at 5 d for human islet cells (a) and 3 d post transduction for rat islet cells (B). Nuclear blue bisbenzimide staining reflects viable cells, whereas red propidium/iodide staining reflects necrotic cells. Magnification, x40.
Effects of IFN- on the morphology and viability of adenovirus-transduced rat and human islet cells
To determine whether the capacity to respond to IFNs is important for islet cell survival during mild adenoviral transduction, we pretreated both rat and human islet cells with 1000 U/ml IFN- for 24 h. Using fluorescence microscopy, we examined rat islet cells at 3 d post transduction and human islet cells at 5 d post transduction with rec700 and pm731.4 viruses and at 7 d post transduction with the recombinant adenoviral vectors. IFN- treatment significantly decreased adenovirus-induced necrosis in both human and rat islet cells to control levels (Fig. 4, A, C, and E). Moreover, the typical distended nuclear morphology induced by adenovirus transduction was significantly reduced in both human and rat islet cells after IFN- treatment (Fig. 4, B and D). To exclude the possibility that IFN- treatment affected adenovirus internalization into islet cells, we analyzed the expression of the 72-kDa DNA binding protein encoded by the E2 region. No effect of IFN- was seen, as assessed by fluorescence microscopic studies (data not shown). These results suggest that type I IFNs are able to induce and/or enhance the transition of pancreatic islet cells to an antiviral state and thereby promote cell survival.
FIG. 4. Effects of IFN- treatment (1000 U/ml) on the viability (A, C, and E) and morphology (B and D) of adenovirus-transduced human (A, B, and E) and rat (C and D) islet cells. Dispersed human and rat islet cells were transduced with various adenoviruses [30 PFU/cell for rat islet cells and 50 PFU/cell for human islet cells when transduced with rec700 and pm734.1 (A–D) and 500 PFU/cell for human islet cells when transduced with dl366, AdCMVProg, and AdTetOFF (E)]. Half the groups were treated for 24 h before and during the transduction with 1000 U/ml IFN- (). Cell viability was assessed on d 5 post transduction (rec700 and pm734.1) or d 7 post transduction (dl366, AdCMVProg, and AdTetOFF) for human islet cells or on d 3 post transduction (rec700 and pm734.1) for rat islet cells, using fluorescence microscopy. At least 200 cells were counted for each group. Results are the mean ± SEM of four (human islet cells) or three (rat islet cells) different experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. nontransduced cells). #, P < 0.05; ##, P < 0.01; ###, P < 0.001 (vs. corresponding groups not treated with IFN-). Statistical significance was determined using a two-way ANOVA and the Bonferroni test.
Evaluation of the antiviral response of rat and human islet cells after adenovirus transduction
The capacity of islet cells to induce an antiviral response as well as the ability of IFN- to modulate this response were investigated by RT-PCR analysis. In particular, we compared the effects of adenovirus exposure and IFN--treatment on the mRNA expression of Ifn- and on the expression of class IA MHC antigen processing and antigen presentation genes as follows: ?2m, Lmp-2, and Tap-1 for human islet cells, and ?2m, Lmp-7, and Tap-1 for rat islet cells.
RT-PCR analysis demonstrated that Ifn- mRNA was expressed under basal conditions by both human and rat islet cells (Fig. 5A). At 24 h post transduction, rec700 significantly decreased the expression of Ifn- mRNA in rat islet cells (Fig. 5A,
), whereas in human islet cells no effect on Ifn- mRNA expression was observed (Fig. 5A, ). Ifn- mRNA expression in human islet cells was not affected by the three deleted adenoviral vectors (Fig. 5E).
FIG. 5. Effects of IFN- treatment and adenoviral exposure on Ifn- (A and E), Lmp-2, Lmp-7 (B and F), Tap-1 (C and G), and ?2m (D and H) mRNA expression in human () and rat () islet cells. After adenoviral transduction [30 PFU/cell for rat islet cells and 50 PFU/cell for human islet cells when transduced with rec700 and pm734.1 (A–D) and 500 PFU/cell for human islet cells when transduced with dl366, AdCMVProg, and AdTetOFF (E–H)] or IFN- (1000 U/ml) treatment, cells were cultured for 24 h in normal RPMI 1640 medium, and total RNA was extracted and analyzed by RT-PCR as described in Materials and Methods. GAPDH or ?-actin (for human islet cells) and ?-actin (for rat islet cells) mRNA expression was assayed as a constitutive control. Results are normalized against nontreated cells and expressed as the mean ± SEM of three different experiments. * and #, P < 0.05; ** and ##, P < 0.01; *** and ###, P < 0.001 [vs. nontreated human (*) or rat (#) cells, using one-way ANOVA and Dunnett’s test].
We also observed basal expression of both low molecular mass polypeptide 7 of the proteasome complex (Lmp-7) for rat islets and Lmp-2 for human islets. However, although in rat islet cells, Lmp-7-mRNA was significantly suppressed by pm734.1 and rec700 (Fig. 5B,), none of the adenoviral constructs affected Lmp-2 expression in human islets (Fig. 5B, , and Fig. 5F).
A similar pattern was observed when we evaluated the expression of transporter associated with antigen processing 1 (Tap-1) mRNA. Adenoviruses (rec700 and pm734.1) induced a clear inhibition in rat islets (Fig. 5C,), whereas in human islets, there was only a nonsignificant trend to lower Tap-1 mRNA in response to AdCMVProg and AdTetOFF (Fig. 5C, , and Fig. 5G).
In both rat and human islet cells, no alteration of ?2m mRNA expression was observed 24 h after adenovirus transduction (Fig. 5D) or transduction with the deleted adenoviral vectors (Fig. 5H). Moreover, in human islet cells, IFN- pretreatment significantly up-regulated mRNA expression of all genes belonging to MHC class IA (?2m, Lmp-2, and Tap-1) and Ifn- mRNA (Fig. 5, E–H). In rat islet cells, IFN- treatment resulted only in up-regulation of Lmp-7-mRNA expression with no other significant alteration of the MHC class IA mRNAs evaluated (Fig. 5,).
Discussion
In a previous study we showed that E1/E3-attenuated adenoviral vectors mediate highly efficient transgene expression. However, the adenovirus-mediated transduction was followed by a significant induction of cellular death, mainly necrosis (12). It is likely that ?-cells located in intact islets are less prone to die in response to adenoviral transduction, because the general viability of free islet cells is diminished compared with that of nondispersed islet cells. Nevertheless, although higher adenoviral titers might be required in the intact islet, the same cell death-inducing events are probably occurring in both cases. Therefore, it is likely that the present results are also relevant to whole islet transduction.
We observed that all adenoviral vectors and adenoviruses, albeit with different efficiencies, induce the death of human and rat islet cells. This is at variance with previous studies in which no adverse effect of adenoviral vectors was observed (3). However, in these studies, intact islets were transduced, which, in our experience, results in transfection of only the cells located at the periphery (Barbu, A., J. Olerud, L. Jansson, M. Welsh, G. Akusj?rvi, and N. Welsh, manuscript in preparation). In addition, in some of these reports, ?-cell function was assessed after only 2–3 d and not after 7 d as in the present investigation. Interestingly, not only are ?-cells negatively affected by adenoviral transduction, but adrenocortical cells have also recently been shown to lose function after transduction with adenoviral vectors (27).
The majority of adenoviral vectors used for gene transfer studies have deletions in their early genes to render the vector replication incompetent. Proteins encoded in these regions are known to induce (E1A and E4-orf 6) or inhibit (E1B, E3, and E4-orf 4) cellular death in transduced host cells (28, 29, 30, 31, 32, 33, 34, 35). Transduction of dispersed rat and human islet cells with dl366 virus, which has intact E1 and E3 regions, but no E4 genes, diminished islet cell viability below the already low concentrations of the viral vector (1 PFU/cell), whereas transduction of islet cells with adenoviruses attenuated by deletions in the E1 and E3 regions (AdCMVProg) promoted cell death to a lesser extent. Moreover, transduction of islet cells with the E1/E3-deleted adenoviral vector, AdTetOFF, which has more comprehensive deletion of E1 and E3 regions than AdCMVProg, resulted in the weakest induction of rat and human islet cell death.
The pronounced cytotoxicity in response to the E4-deleted adenoviral vector is not surprising considering that vectors with intact E1 regions have higher transcription of adenoviral genes than vectors without E1 (36). Thus, there is probably a larger de novo synthesis of viral proteins from the E4-deleted than from the E1/E3-deleted vector. Indeed, we observed high ADP and pIX mRNA levels in dl366-transduced cells compared with AdCMVProg-transduced cells.
Our observations also point out the possibility that the E3 gene product ADP and the E1 located adenoviral protein IX, which are usually expressed at late stages of transduction, might participate in adenovirus-induced islet cell death. The expression of genes located in the E3 and E4 regions of the adenoviral genome is usually low in the absence of E1A genes (37). However, other studies have shown residual expression of E4 genes in cells transduced with E1A-deleted vectors (35). In line with these results, we presently demonstrate residual expression of two genes of the adenoviral genome ADP and the adenoviral gene IX in cells transduced with AdCMVProg. This basal residual expression seems to be sufficient for modulating cell death in islet cells.
To explore the effect of adenovirus death protein on islet cell viability, we transduced rat and human islet cells with adenoviruses with differential expression of ADP. As expected, both adenoviruses (rec700 and pm734.1) were potent and rapid inducers of islet cell death (2–3 d for rat cells and 4–5 d for human cells), and the adenovirus expressing ADP (rec700) induced cell death more potently than the vector lacking ADP (pm734.1). Thus, the expression of ADP negatively affects the viability of adenovirus-transduced islet cells.
As in our previous study (12), adenovirus transduction of islet cells resulted mostly in islet cell necrosis, which was paralleled by a distended morphology of the nuclei. This feature of the adenoviral transduction was previously reported and might be related to the capacity of the E1B-19K protein to alter the organization of the intermediary filaments and nuclear lamina in transduced cells (38). The fact that recombinant adenoviral vectors with intact E1 regions (dl366), but not recombinant adenoviral vectors with E1 deletions (AdCMVProg), induce a morphology (results not shown) similar to that observed with the rec700 and pm734.1 adenoviruses also supports this hypothesis.
As discussed above, our results support a modulating role of ADP in adenovirus-mediated islet cell death. In addition, it is possible that the product of the adenoviral gene IX, pIX, could enhance this cytotoxic effect. The adenovirus protein pIX is expressed after the early adenoviral genes and has been shown to be incorporated into the viral capsid, where it is in part responsible for virion stability. More recently, pIX has been identified as a transcriptional activator (39) with the capacity to enhance gene expression from E1A, E4, and the major late adenoviral promoters. With this function, pIX might very well be able to modulate death pathways in targeted cells. However, in the few studies addressing this issue, its capacity to enhance gene transcription varies considerably (from 1.4- to 70-fold increase in gene expression) (39, 40) and may be dependent on the cellular system studied. Therefore, the specific effects of IX+/– recombinant adenoviral vectors on the viability of transduced islet cells should be investigated.
From another perspective, the study of islet cell-adenovirus/recombinant adenovirus interactions can reveal important features concerning the pathogenesis of type 1 diabetes, because viral transduction, although mostly of enteroviral origin, has been implicated as an important environmental factor that may trigger the subsequent autoimmune reaction against ?-cells in genetically susceptible individuals (41, 42). In this context, two scenarios have been proposed: 1) the ?-cell defense against viral transduction is in some cases low, which results in ?-cell necrosis and also autoimmunity (43); and 2) the ?-cell IFN response is strong, which leads to the survival of the ?-cell after viral transduction, but also to autoimmunity (44). The second hypothesis is supported by the finding that ?-cells may constitutively express high levels of IFN- (44).
Type I IFNs (IFN-/?) are produced very rapidly (within hours) in direct response to viral transductions, and they are believed to strongly induce the antiviral state in target cells (45). In our system, however, the IFN response in pancreatic islets was not triggered by adenoviral transduction or adenoviral vectors. RT-PCR studies of mRNA expression of the Ifn- and MHC class IA genes revealed no major effect of virus exposure on human islets, whereas in rat cells a significant down-regulation of MHC class IA gene expression was observed, consistent with down-regulation of the Ifn- gene. These data suggest that pancreatic islet cells are not able to build up an antiviral state after adenoviral transduction and/or that the adenoviral proteins (E1A) are very efficient blockers of the IFN signal transduction pathway (46) in this type of cell. Furthermore, in our experience, exogenous addition of IFN- was vital for preventing adenovirus-induced islet cell death. Indeed, treatment of rat and human pancreatic islets with IFN- before and during transduction blocked adenovirus-induced cytolysis and prevented the nuclear structural modifications of the transduced cells. The protective effect of type I IFNs may be related to their capacity to suppress virus replication and inhibit early viral gene expression (47). The ultimate outcome of IFN signaling is activation of the transcription of target genes, such as 2',5'-oligoadenylate synthetase, MxA, MxB genes (48) and MHC class IA antigens (16, 17). Consistent with these reports, we found that IFN- treatment was able to up-regulate expression of MHC class IA genes (?2m, Lmp-2, and Tap-1) in human islets. In rat islet cells, exogenous addition of IFN- stimulated expression of the proteasome subunit Lmp-7 and ?2m mRNA, although the later was not statistically significant. Thus, these combined observations are not compatible with the second scenario, described in the previous paragraph, stating that the ?-cell IFN response is strong. Instead, they support the idea that the virus-induced ?-cell IFN response is suboptimal, leading to ?-cell death and possibly autoimmune triggering. It may be that the pancreatic ?-cell is exceptional in this aspect, because other cell types are known to induce an IFN response when transduced with adenovirus (49, 50).
Previous studies with isolated pancreatic islets have revealed that human ?-cells are clearly more resistant against toxin- and cytokine-induced damage than rodent cells (51, 52). Our present results suggest that this is also the case in adenovirus-induced islet cell death. Moreover, in response to IFN- treatment, human islet cells seem to be more efficient in inducing genes belonging to the antigen processing and antigen presentation MHC class IA family, which might be a critical event in the context of viral transduction.
Genetic engineering of ?-cells for use in gene therapy approaches aims at enhancing the resistance against poor grafting, rejection, and autoimmune attack in type 1 diabetes. For this purpose as well as for basic ?-cell research, viral-derived gene transfer tools should not have any impact by themselves on the function, morphology, or viability of target cells. We showed in this study that prolonged culture of adenoviral-transduced islet cells leads to significant cytotoxicity and that pancreatic islet cells do not have the capacity to induce an IFN response. This process is more evident when high concentrations of the virus are used, and the outcome seems to depend upon the extent of the deletion of the viral genome. If inappropriate viral vectors are chosen in an in vivo situation, pancreatic ?-cells could be directly destroyed by adenovirus-induced cytolysis, or alternatively, the transduction process could cause an inflammatory reaction, leading to or exacerbating the ?-cell-targeted autoimmune response. Considering the wide use of adenoviral vehicles for genetic modification of nondividing cells, these findings should be considered in the design and application of such vectors in experimental and clinical ?-cell research.
Acknowledgments
We gratefully acknowledge the excellent technical assistance of Ing-Marie M?rsare. We also thank Prof. William S. M. Wold (Department of Molecular Microbiology and Immunology, St. Louis University School of Medicine, St. Louis, MO) for providing us with the wild-type adenoviruses.
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Address all correspondence and requests for reprints to: Dr. Andreea Barbu, Department of Medical Cell Biology, Biomedical Center, P.O. Box 571, S-751 23 Uppsala, Sweden. E-mail: andreea.barbu@medcellbiol.uu.se.
Abstract
As adenoviral vectors are extensively used for genetic manipulation of insulin-producing cells in vitro, there is an increasing need to evaluate their effects on the function, morphology, and viability of transduced pancreatic islets. In the present study we observed that specific adenoviral genotypes, carrying E4 and E1/E3 deletions, correlate with differential induction of necrosis in pancreatic islet cells. In particular, the adenovirus death protein encoded from the E3 region of the adenoviral genome was able to modulate the changes induced in the morphology and viability of the transduced cells. We also propose a putative role for the transcriptional regulator pIX. Although human islet cells showed an increased resistance in terms of viral concentrations required for the induction of cell toxicity, our results showed that they were unable to build up an efficient antiviral response after transduction and that their survival was dependent on the exogenous addition of -interferon. An intact and fully functional ?-cell is crucial for the successful application of gene therapy approaches in type 1 diabetes, and therefore, the implications of our findings need to be considered when designing vectors for gene transfer into pancreatic ?-cells.
Introduction
TYPE 1 DIABETES IS an autoimmune disease that eventually causes complete destruction of insulin-secreting ?-cells in the pancreas. Recently, pancreatic islet transplantation has been validated as a promising alternative to correct for the insulin deficiency in type 1 diabetes (1). However, the requirement for islets derived from two to four donors to treat a single patient is a barrier to the use of this technique on a larger scale. As a result, much effort is going into developing new therapeutic strategies to increase durable functional islet mass. Gene therapy offers a potential means to protect against ?-cell death by induction of immunoregulatory, cytoprotective, or antiapoptotic genes (2, 3) as well as to stimulate ?-cell differentiation and regeneration by induction of different transcriptional regulators and growth factors (4, 5, 6).
The ability to engineer pancreatic ?-cells is a prerequisite for successful application of gene therapy approaches in type 1 diabetes. However, pancreatic islets are terminally differentiated cell clusters that are difficult to reach in vivo and troublesome to maintain in tissue culture. Therefore, the use of nonviral transfection strategies for genetic modification of insulin-producing cells, including lipofection, electroporation, and biolistic particles, mediate only low transfection efficiencies (7, 8). In contrast, due to their capacity to mediate highly efficient gene transfer in nondividing cells, viral vectors have emerged as the first choice for engineering ?-cells. Together with lentiviruses (9) and adeno-associated viruses (10), adenoviruses are the most commonly used viral vectors in current ?-cell research (11, 12). However, despite their advantages, such as high titer production and the diminished risk for insertional mutagenesis due to the epichromosomal location within the target cell, adenoviral transduction and gene transfer may interfere with ?-cell function and/or induce cell death. Adenoviruses contain genes that are able to initiate and modulate cell death by inducing or suppressing specific processes within the target cell, and these effects are likely to be dependent on both the genotype of the viral vector used and the cell system studied (13). Adenoviral genes are transcribed in a complex temporal manner. Early genes are transcribed from five different promoters, in the order E1A, E4, E3, E1B, and E2. These genes are mainly concerned with the regulation of viral gene expression and DNA replication and the modulation of cell death. The E3–11.6K protein, also known as the adenovirus death protein (ADP), is encoded by the E3 transcription unit of the adenoviral genome, but its expression is turned on preferentially by a strong adenovirus major late promoter. In contrast to the other antiapoptotic genes located in the same region, ADP has been shown to facilitate viral exit from the nucleus of the infected cells by inducing cell death (14). The early production of interferons (IFNs) is believed to be an important host response to many viral infections, including adenoviruses (15), and, in pancreatic islets, IFN production has been associated with hyperexpression of major histocompatibility complex (MHC) class IA antigens (16, 17).
Previously, we reported that E1/E3-deleted adenoviral vectors induce ?-cell cytotoxicity (12). In the present study we investigate the effects of various adenoviral genome deletions on rat and human islet cell viability to determine whether a specific adenoviral genotype correlates with ?-cell death. We also try to determine whether islet cells are able to establish an antiviral state in response to adenoviral transduction and the role of type I IFNs during this process.
Materials and Methods
Reagents
RPMI 1640, fetal calf serum (FCS), L-glutamine, penicillin/streptomycin, trypsin-EDTA, bisbenzimide (Hoechst-33342), and propidium iodide were purchased from Sigma-Aldrich Corp. (St. Louis, MO). All other reagents used, unless otherwise specified, were obtained from E. Merck (Darmstadt, Germany).
Cell culture
Rat pancreatic islets of Langerhans were isolated from 3-month-old Sprague Dawley rats (local Uppsala colony) by a collagenase digestion procedure and cultured in groups of 150/50-mm well in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, benzylpenicillin (100 U/ml), and streptomycin (0.1 mg/ml), containing 11.1 mM glucose (18). Human pancreatic islets, provided by Prof. Olle Korsgren (Uppsala University, Uppsala, Sweden), were cultured in the medium described above, containing 5.6 mM glucose, at 37 C in humidified air with 5% CO2. Culture medium was replaced every 48 h. The use of rat and human pancreatic islets was in accordance with international guidelines (NIH publication 85-23, revised 1985) and was approved by the local ethics committees for Uppsala University.
Adenoviral vectors and viruses
The following human adenovirus (Ad) serotype 5 mutants were used in this study: 1) AdCMVProg, from a progesterone-inducible system, in which the cytomegalovirus (CMV) promoter drives the cDNA for an inactive transactivator protein (genotype E1/E3-deleted 455–3328 and 30005–30750) (19); 2) Adeno-X-Tet-Off (BD Clontech Laboratories, Inc., Palo Alto, CA), encoding a tetracycline-controlled transactivator protein (genotype E1/E3-deleted, 342–3528 and 27865–30995, with bigger deletions both in the E1 region, encompassing a portion of the pIX promotor, and in the E3 region of the adenoviral genome, encompassing ADP) (20); and 3) dl366, E4-deleted, expressing none of the products of the early region 4 (21). The Adeno-X-Tet-Off viral vector is similar to the AdEasy-1 vector, but not identical, because AdEasy-1 is deleted in nucleotides 480-3533 and 28130–30820 (22). All virus stocks were purified by cesium chloride density gradient centrifugation (L-80 ultracentrifuge, Beckman Coulter, Fullerton, CA) and plaque-titrated by serial dilution and agar overlay on their respective transcomplementing cell lines. Typical titers were 109 plaque-forming units (PFU)/ml or higher, representing 1–5% of the total viral particles as determined by readings of the ODs.
The E3-ADP variant adenoviruses used in this study, rec700 and pm734.1, were provided by Prof. William S. M. Wold (St. Louis University School of Medicine, St. Louis, MO). rec700 is an Ad5-Ad2-Ad5 recombinant (23) and is the parental virus for pm734.1 (with ADP residues 1–48 deleted) (14).
Adenoviral transduction, viral transduction, and assessment of transfection efficiency
Rat and human islets were dispersed into individual cells by treatment with trypsin (5 mg/ml) for 5 min, in Ca2+- and Mg2+-free Hanks’ solution and were cultured for an additional 1 h at 37 C. Single cells from dispersed islets were transduced with adenoviral vectors or wild-type viruses in a minimum volume of 0.1 ml RPMI 1640 supplemented with 2% FCS, containing various adenoviral concentrations (PFU per cell). After 1-h incubation at 37 C, islet cells were washed with RPMI 1640 medium and plated in 96- or 24-well plates, coated with 0.1% gelatin. The plates were incubated at 37 C, and medium was changed every second day for up to 1 wk post transduction. In some cases, 24 h before transduction and during the experiments, islet cells were treated with 1000 U/ml rat or human IFN- (PBL Biomedical Laboratories, Piscataway, NJ).
To assess the efficiency of the adenoviral-induced ?-cell transduction, we coincubated intact islets and dispersed rat islet cells with 100 PFU/cell of an adenoviral vector expressing the ?-galactosidase gene under control of the CMV promoter. The next day islets were dispersed, cells were fixed, and enzymatic activity and transduction efficiency were determined using light microscopy studies as previously described (24). To evaluate the capacity of wild-type adenoviruses to infect dispersed islet cells, we performed fluorescence microscopy studies after staining for the E2–72kD adenovirus DNA binding protein (25).
Sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT) assay
Adenoviral-induced cell death was assessed using the colorimetric assay (XTT-based) for cell viability (12). Adenoviral-transduced and nontransduced rat and human islet cells were plated in 96-well plates in 100 μl complete RPMI 1640 culture medium. Three days after transduction when using E3-ADP variant adenoviruses or 7 d after transduction when using recombinant adenoviral vectors, 50 μl 1 mg/ml XTT (Roche, Mannheim, Germany) was added to each well. After 5 h of incubation at 37 C, the ODs were read in a Microplate autoreader (Bio-Tek Instruments, Inc., Watford, UK) at 490 nm.
Fluorescence microscopy
To investigate the effect of adenoviruses on islet cell viability, we performed fluorescence microscopy studies after 3–7 d (rat islet cells) and 5–7 d (human islet cells) of culture post transduction. Rat and human islet cells were incubated in RPMI 1640 complete medium containing 5 μg/ml bisbenzimide and 10 μg/ml propidium iodide for 10 min at 37 C. The cells were then washed, detached by mild trypsination, and examined by fluorescence microscopy.
RNA extraction and cDNA synthesis
Twenty to 24 h after transduction of rat and human islets with adenoviruses, total cellular RNA was isolated by a single-step method, using a commercial system (Ultraspec RNA isolation system, Biotecx Laboratories, Inc., Houston, TX), followed by digestion with ribonuclease-free deoxyribonuclease (Qiagen, Chatsford, CA). The total RNA for each sample was reverse transcribed with Moloney murine leukemia virus reverse transcriptase using an oligo(deoxythymidine) primer in the presence of 5 mM MgCl2, 1 mM of each deoxy-NTP, and 1 U/μl recombinant RNasin ribonuclease inhibitor. The RT reaction was performed at 42 C for 40 min, followed by inactivation of the enzyme at 95 C for 5 min.
Real-time PCR analysis
On the basis of the respective rat and human cDNAs, primers were designed to amplify specific regions of Ifn-, class IA MHC antigen-processing and antigen presentation genes [?2-microglobulin (?2m), low molecular mass polypeptide 2 (Lmp-2), and transporter associated with antigen processing 1 (Tap-1) for human islet cells; ?2m, Lmp-7, and Tap-1 for rat islet cells], and the ?-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs. PCR amplifications were carried out in a real time LightCycler (Roche) using the temperature-time sequence: denaturation for 15 sec at 95 C, annealing for 13 sec at 55 C, and extension for 6 sec at 72 C, and the primer sequences described in Table 1.
TABLE 1. PCR primers used to assess gene expression in human and rat islet cells
To obtain relative mRNA expression values, PCRs were performed with ?-actin for rat samples and GAPDH or ?-actin for human samples for each of the experimental groups. The amount of PCR product relative to these housekeeping genes was calculated as previously described (26). For each experiment the data are presented as normalized values against the relative mRNA levels of the control samples. All PCR products were verified by agarose gel electrophoresis and SYBR Gold (Molecular Probes, Eugene, OR) staining.
Statistical analysis
Data are summarized as the mean ± SEM. The significances of differences between the groups were determined by one- or two-way ANOVA for repeated measurements and the Bonferroni or Dunnett’s test. Differences were considered significant at P < 0.05. Statistical analysis was performed using SigmaStat (SPSS Science Software, Erkrath, Germany).
Results
To evaluate the efficacy of adenoviral vectors to transduce islet cells, we stained for ?-galactosidase activity and counted cells in a light microscope. In a typical experiment we observed more than 90% ?-galactosidase-positive cells in the dispersed islet cell group and only 20–30% ?-galactosidase-positive cells in the intact islet cell group (results not shown). In all subsequent transduction experiments, only dispersed islet cells were used to ensure high transduction efficiency. The adenoviral vector AdCMVProg belongs to an inducible system that we have previously described (12). We showed that increasing concentrations of the virus resulted in a corresponding increase in protein expression levels, with more than 70% ?-cells transduced at 100 PFU/cell. We obtained similar transduction efficiencies (>80%) with another adenoviral vector expressing green fluorescent protein that has the same backbone as the AdTetOFF vector used in the present study.
Effects of different adenoviral vector genomes on rat and human islet cell viability
To determine whether the adenoviral-induced cytotoxicity correlates to a particular adenoviral genotype, we transduced dispersed rat and human islet cells with different replication deficient adenoviral vectors carrying deletions in one or more of the E1, E3, or E4 regions of the adenoviral genome.
XTT-based cell viability assays performed 7 d after adenoviral vector/islet cell incubation, revealed that transduction of rat islet cells with dl366 virus (carrying intact E1 and E3 regions, but none of the genes of the E4 region) significantly diminished islet cell viability (to 61.8 ± 0.038%) at 1 PFU/cell and that the adenoviral-induced cell death occurred in a dose-dependent manner (with no viable cells left at 100 PFU/cell; Fig. 1A,
). To investigate whether this cytotoxicity was due to nonspecific effects of proteins belonging to the adenoviral capsids we coincubated islet cells with heat-inactivated dl366 viruses. One week post transduction, no effect on the cell viability was observed (results not shown), suggesting that adenovirus-induced cell death is secondary to the internalization of functional vector particles into the cells. Adenovirus-mediated toxicity was also seen after transduction of islet cells with the E1/E3-attenuated viral vector, AdCMVProg (Fig. 1B,
), although to a lesser extent. Thus, 1–10 PFU/cell of the E4 dl366 virus induced 50% cytotoxicity in rat islet cells, whereas in AdCMVProg-transduced cells the same effect was obtained only at concentrations as high as 10–100 PFU/cell. Interestingly, transduction of rat islet cells with AdTetOFF vector, another E1/E3-deleted viral vector that carries additional deletions in the E1 region (encompassing a portion of the pIX promotor) and in the E3 region (encompassing ADP), resulted in a lesser induction of islet cell death and only at a high concentration of the vector (Fig. 1C,
).
FIG. 1. Decreased islet cell viability in response to dl366 (A), AdCMVProg (B), and AdTetOFF (C) adenoviral transduction. Dispersed human () and rat () islet cells were transduced with various concentrations of the adenoviral vectors, and cellular viability was assessed 7 d post transduction using an XTT-based assay. Individual colorimetric readings are normalized to the value of nontransduced islet cells. Results are the mean ± SEM of eight (rat islet cells) or 10 (human islet cells) separate experiments. * and #, P < 0.05; ** and ##, P < 0.01; *** and ###, P < 0.001 [vs. nontransduced human (*) or rat (#) cells, by one-way ANOVA, Bonferroni test]. D, ADP and pIX mRNA expression in rat and human islet cells after adenoviral exposure was analyzed by RT-PCR as described in Materials and Methods. The reaction was stopped during the exponential phase (23 cycles for ADP and pIX and 20 cycles for ?-actin), and the products were analyzed by ethidium bromide staining and agarose gel electrophoresis. E, Schematic of the adenoviral genome and the specific deletions of the adenoviral vectors used in this study: E1–E4, early transcribed adenoviral genes; MLP, major late promotor; VA-RNA, adenoviral RNA.
In human islet cells, transduction with dl366 vector induced a similar pattern of cytotoxicity, but at considerably higher viral concentrations (100 PFU/cell reduced cellular viability to 55.3 ± 10.9% in human islet cells vs. 0% in rat islet cells; Fig 1A, ). At high concentrations (100 PFU/cell) both E1/E3-attenuated adenoviruses reduced islet cell viability to 89.4 ± 3.9% when cells were transduced with AdTetOFF (Fig. 1C, ) and 54 ± 13.3% when cells were transduced with AdCMVProg (Fig. 1B, ). These results are in line with our previous observation that an adenoviral vector induces cytotoxicity in pancreatic islet cells (12) and suggest that this effect is modulated by the adenoviral genome and the particular characteristics of the target cells.
Effects of ADP on wild-type adenovirus-induced islet cell toxicity
RT-PCR analysis performed on mRNA isolated from transduced islet cells demonstrated a differential expression of the adenoviral genes IX and ADP. As anticipated, we could not detect ADP and IX cDNA in control or AdTetOFF cells (Fig. 1D). In addition, expression of the ADP and IX genes was clearly stronger in cells transduced with dl366 than in those transduced with AdCMVProg (Fig. 1D). Taken together, these data (Fig. 1) raise the possibility that the cytotoxic effect of adenoviruses in islet cells may be in part correlated to ADP expression. Indeed, after transduction of rat islet cells with an ADP– virus (pm734.1), we observed a milder effect on islet cell viability, as assessed by the XTT-based metabolic assay (Fig. 2A). Although not statistically significant, there was also a trend for a milder effect of the ADP– virus in human islet cells (Fig. 2B).
FIG. 2. Estimation of cellular cytotoxicity induced in dispersed islet cells by adenoviruses carrying differential expression of ADP. Rat islet cells (A) and human islet cells (B) were transduced with various concentrations of rec700 or pm734.1 virus. Cell viability was assessed on d 3 (rat islet cells) or d 5 post transduction (human islet cells) using the XTT-based assay. Individual colorimetric readings are normalized to the value of nontransduced islet cells. Results are the mean ± SEM of six separate experiments. Statistical significance was determined by two-way ANOVA and Dunnett’s test.
Fluorescence microscopic analysis after double staining with bisbenzimide/propidium iodide indicated that viral exposure promoted necrosis in both human (Fig. 3A) and rat (Fig. 3B) islet cells. The nuclei of the transduced cells were extremely distended, and this specific morphology preceded the rupture of the plasma membrane. Note that no increase in typical apoptotic features was observed in the transduced cells.
FIG. 3. Micrographs showing bisbenzimide/propidium iodide fluorescence of human (A) and rat (B) islet cells. Cells were transduced with 30 PFU/cell (rat cells) or 50 PFU/cell (human cells) of rec700 (wild type) and pm734.1 (ADP–), and cell morphology was evaluated by fluorescence microscopy at 5 d for human islet cells (a) and 3 d post transduction for rat islet cells (B). Nuclear blue bisbenzimide staining reflects viable cells, whereas red propidium/iodide staining reflects necrotic cells. Magnification, x40.
Effects of IFN- on the morphology and viability of adenovirus-transduced rat and human islet cells
To determine whether the capacity to respond to IFNs is important for islet cell survival during mild adenoviral transduction, we pretreated both rat and human islet cells with 1000 U/ml IFN- for 24 h. Using fluorescence microscopy, we examined rat islet cells at 3 d post transduction and human islet cells at 5 d post transduction with rec700 and pm731.4 viruses and at 7 d post transduction with the recombinant adenoviral vectors. IFN- treatment significantly decreased adenovirus-induced necrosis in both human and rat islet cells to control levels (Fig. 4, A, C, and E). Moreover, the typical distended nuclear morphology induced by adenovirus transduction was significantly reduced in both human and rat islet cells after IFN- treatment (Fig. 4, B and D). To exclude the possibility that IFN- treatment affected adenovirus internalization into islet cells, we analyzed the expression of the 72-kDa DNA binding protein encoded by the E2 region. No effect of IFN- was seen, as assessed by fluorescence microscopic studies (data not shown). These results suggest that type I IFNs are able to induce and/or enhance the transition of pancreatic islet cells to an antiviral state and thereby promote cell survival.
FIG. 4. Effects of IFN- treatment (1000 U/ml) on the viability (A, C, and E) and morphology (B and D) of adenovirus-transduced human (A, B, and E) and rat (C and D) islet cells. Dispersed human and rat islet cells were transduced with various adenoviruses [30 PFU/cell for rat islet cells and 50 PFU/cell for human islet cells when transduced with rec700 and pm734.1 (A–D) and 500 PFU/cell for human islet cells when transduced with dl366, AdCMVProg, and AdTetOFF (E)]. Half the groups were treated for 24 h before and during the transduction with 1000 U/ml IFN- (). Cell viability was assessed on d 5 post transduction (rec700 and pm734.1) or d 7 post transduction (dl366, AdCMVProg, and AdTetOFF) for human islet cells or on d 3 post transduction (rec700 and pm734.1) for rat islet cells, using fluorescence microscopy. At least 200 cells were counted for each group. Results are the mean ± SEM of four (human islet cells) or three (rat islet cells) different experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. nontransduced cells). #, P < 0.05; ##, P < 0.01; ###, P < 0.001 (vs. corresponding groups not treated with IFN-). Statistical significance was determined using a two-way ANOVA and the Bonferroni test.
Evaluation of the antiviral response of rat and human islet cells after adenovirus transduction
The capacity of islet cells to induce an antiviral response as well as the ability of IFN- to modulate this response were investigated by RT-PCR analysis. In particular, we compared the effects of adenovirus exposure and IFN--treatment on the mRNA expression of Ifn- and on the expression of class IA MHC antigen processing and antigen presentation genes as follows: ?2m, Lmp-2, and Tap-1 for human islet cells, and ?2m, Lmp-7, and Tap-1 for rat islet cells.
RT-PCR analysis demonstrated that Ifn- mRNA was expressed under basal conditions by both human and rat islet cells (Fig. 5A). At 24 h post transduction, rec700 significantly decreased the expression of Ifn- mRNA in rat islet cells (Fig. 5A,
), whereas in human islet cells no effect on Ifn- mRNA expression was observed (Fig. 5A, ). Ifn- mRNA expression in human islet cells was not affected by the three deleted adenoviral vectors (Fig. 5E).
FIG. 5. Effects of IFN- treatment and adenoviral exposure on Ifn- (A and E), Lmp-2, Lmp-7 (B and F), Tap-1 (C and G), and ?2m (D and H) mRNA expression in human () and rat () islet cells. After adenoviral transduction [30 PFU/cell for rat islet cells and 50 PFU/cell for human islet cells when transduced with rec700 and pm734.1 (A–D) and 500 PFU/cell for human islet cells when transduced with dl366, AdCMVProg, and AdTetOFF (E–H)] or IFN- (1000 U/ml) treatment, cells were cultured for 24 h in normal RPMI 1640 medium, and total RNA was extracted and analyzed by RT-PCR as described in Materials and Methods. GAPDH or ?-actin (for human islet cells) and ?-actin (for rat islet cells) mRNA expression was assayed as a constitutive control. Results are normalized against nontreated cells and expressed as the mean ± SEM of three different experiments. * and #, P < 0.05; ** and ##, P < 0.01; *** and ###, P < 0.001 [vs. nontreated human (*) or rat (#) cells, using one-way ANOVA and Dunnett’s test].
We also observed basal expression of both low molecular mass polypeptide 7 of the proteasome complex (Lmp-7) for rat islets and Lmp-2 for human islets. However, although in rat islet cells, Lmp-7-mRNA was significantly suppressed by pm734.1 and rec700 (Fig. 5B,), none of the adenoviral constructs affected Lmp-2 expression in human islets (Fig. 5B, , and Fig. 5F).
A similar pattern was observed when we evaluated the expression of transporter associated with antigen processing 1 (Tap-1) mRNA. Adenoviruses (rec700 and pm734.1) induced a clear inhibition in rat islets (Fig. 5C,), whereas in human islets, there was only a nonsignificant trend to lower Tap-1 mRNA in response to AdCMVProg and AdTetOFF (Fig. 5C, , and Fig. 5G).
In both rat and human islet cells, no alteration of ?2m mRNA expression was observed 24 h after adenovirus transduction (Fig. 5D) or transduction with the deleted adenoviral vectors (Fig. 5H). Moreover, in human islet cells, IFN- pretreatment significantly up-regulated mRNA expression of all genes belonging to MHC class IA (?2m, Lmp-2, and Tap-1) and Ifn- mRNA (Fig. 5, E–H). In rat islet cells, IFN- treatment resulted only in up-regulation of Lmp-7-mRNA expression with no other significant alteration of the MHC class IA mRNAs evaluated (Fig. 5,).
Discussion
In a previous study we showed that E1/E3-attenuated adenoviral vectors mediate highly efficient transgene expression. However, the adenovirus-mediated transduction was followed by a significant induction of cellular death, mainly necrosis (12). It is likely that ?-cells located in intact islets are less prone to die in response to adenoviral transduction, because the general viability of free islet cells is diminished compared with that of nondispersed islet cells. Nevertheless, although higher adenoviral titers might be required in the intact islet, the same cell death-inducing events are probably occurring in both cases. Therefore, it is likely that the present results are also relevant to whole islet transduction.
We observed that all adenoviral vectors and adenoviruses, albeit with different efficiencies, induce the death of human and rat islet cells. This is at variance with previous studies in which no adverse effect of adenoviral vectors was observed (3). However, in these studies, intact islets were transduced, which, in our experience, results in transfection of only the cells located at the periphery (Barbu, A., J. Olerud, L. Jansson, M. Welsh, G. Akusj?rvi, and N. Welsh, manuscript in preparation). In addition, in some of these reports, ?-cell function was assessed after only 2–3 d and not after 7 d as in the present investigation. Interestingly, not only are ?-cells negatively affected by adenoviral transduction, but adrenocortical cells have also recently been shown to lose function after transduction with adenoviral vectors (27).
The majority of adenoviral vectors used for gene transfer studies have deletions in their early genes to render the vector replication incompetent. Proteins encoded in these regions are known to induce (E1A and E4-orf 6) or inhibit (E1B, E3, and E4-orf 4) cellular death in transduced host cells (28, 29, 30, 31, 32, 33, 34, 35). Transduction of dispersed rat and human islet cells with dl366 virus, which has intact E1 and E3 regions, but no E4 genes, diminished islet cell viability below the already low concentrations of the viral vector (1 PFU/cell), whereas transduction of islet cells with adenoviruses attenuated by deletions in the E1 and E3 regions (AdCMVProg) promoted cell death to a lesser extent. Moreover, transduction of islet cells with the E1/E3-deleted adenoviral vector, AdTetOFF, which has more comprehensive deletion of E1 and E3 regions than AdCMVProg, resulted in the weakest induction of rat and human islet cell death.
The pronounced cytotoxicity in response to the E4-deleted adenoviral vector is not surprising considering that vectors with intact E1 regions have higher transcription of adenoviral genes than vectors without E1 (36). Thus, there is probably a larger de novo synthesis of viral proteins from the E4-deleted than from the E1/E3-deleted vector. Indeed, we observed high ADP and pIX mRNA levels in dl366-transduced cells compared with AdCMVProg-transduced cells.
Our observations also point out the possibility that the E3 gene product ADP and the E1 located adenoviral protein IX, which are usually expressed at late stages of transduction, might participate in adenovirus-induced islet cell death. The expression of genes located in the E3 and E4 regions of the adenoviral genome is usually low in the absence of E1A genes (37). However, other studies have shown residual expression of E4 genes in cells transduced with E1A-deleted vectors (35). In line with these results, we presently demonstrate residual expression of two genes of the adenoviral genome ADP and the adenoviral gene IX in cells transduced with AdCMVProg. This basal residual expression seems to be sufficient for modulating cell death in islet cells.
To explore the effect of adenovirus death protein on islet cell viability, we transduced rat and human islet cells with adenoviruses with differential expression of ADP. As expected, both adenoviruses (rec700 and pm734.1) were potent and rapid inducers of islet cell death (2–3 d for rat cells and 4–5 d for human cells), and the adenovirus expressing ADP (rec700) induced cell death more potently than the vector lacking ADP (pm734.1). Thus, the expression of ADP negatively affects the viability of adenovirus-transduced islet cells.
As in our previous study (12), adenovirus transduction of islet cells resulted mostly in islet cell necrosis, which was paralleled by a distended morphology of the nuclei. This feature of the adenoviral transduction was previously reported and might be related to the capacity of the E1B-19K protein to alter the organization of the intermediary filaments and nuclear lamina in transduced cells (38). The fact that recombinant adenoviral vectors with intact E1 regions (dl366), but not recombinant adenoviral vectors with E1 deletions (AdCMVProg), induce a morphology (results not shown) similar to that observed with the rec700 and pm734.1 adenoviruses also supports this hypothesis.
As discussed above, our results support a modulating role of ADP in adenovirus-mediated islet cell death. In addition, it is possible that the product of the adenoviral gene IX, pIX, could enhance this cytotoxic effect. The adenovirus protein pIX is expressed after the early adenoviral genes and has been shown to be incorporated into the viral capsid, where it is in part responsible for virion stability. More recently, pIX has been identified as a transcriptional activator (39) with the capacity to enhance gene expression from E1A, E4, and the major late adenoviral promoters. With this function, pIX might very well be able to modulate death pathways in targeted cells. However, in the few studies addressing this issue, its capacity to enhance gene transcription varies considerably (from 1.4- to 70-fold increase in gene expression) (39, 40) and may be dependent on the cellular system studied. Therefore, the specific effects of IX+/– recombinant adenoviral vectors on the viability of transduced islet cells should be investigated.
From another perspective, the study of islet cell-adenovirus/recombinant adenovirus interactions can reveal important features concerning the pathogenesis of type 1 diabetes, because viral transduction, although mostly of enteroviral origin, has been implicated as an important environmental factor that may trigger the subsequent autoimmune reaction against ?-cells in genetically susceptible individuals (41, 42). In this context, two scenarios have been proposed: 1) the ?-cell defense against viral transduction is in some cases low, which results in ?-cell necrosis and also autoimmunity (43); and 2) the ?-cell IFN response is strong, which leads to the survival of the ?-cell after viral transduction, but also to autoimmunity (44). The second hypothesis is supported by the finding that ?-cells may constitutively express high levels of IFN- (44).
Type I IFNs (IFN-/?) are produced very rapidly (within hours) in direct response to viral transductions, and they are believed to strongly induce the antiviral state in target cells (45). In our system, however, the IFN response in pancreatic islets was not triggered by adenoviral transduction or adenoviral vectors. RT-PCR studies of mRNA expression of the Ifn- and MHC class IA genes revealed no major effect of virus exposure on human islets, whereas in rat cells a significant down-regulation of MHC class IA gene expression was observed, consistent with down-regulation of the Ifn- gene. These data suggest that pancreatic islet cells are not able to build up an antiviral state after adenoviral transduction and/or that the adenoviral proteins (E1A) are very efficient blockers of the IFN signal transduction pathway (46) in this type of cell. Furthermore, in our experience, exogenous addition of IFN- was vital for preventing adenovirus-induced islet cell death. Indeed, treatment of rat and human pancreatic islets with IFN- before and during transduction blocked adenovirus-induced cytolysis and prevented the nuclear structural modifications of the transduced cells. The protective effect of type I IFNs may be related to their capacity to suppress virus replication and inhibit early viral gene expression (47). The ultimate outcome of IFN signaling is activation of the transcription of target genes, such as 2',5'-oligoadenylate synthetase, MxA, MxB genes (48) and MHC class IA antigens (16, 17). Consistent with these reports, we found that IFN- treatment was able to up-regulate expression of MHC class IA genes (?2m, Lmp-2, and Tap-1) in human islets. In rat islet cells, exogenous addition of IFN- stimulated expression of the proteasome subunit Lmp-7 and ?2m mRNA, although the later was not statistically significant. Thus, these combined observations are not compatible with the second scenario, described in the previous paragraph, stating that the ?-cell IFN response is strong. Instead, they support the idea that the virus-induced ?-cell IFN response is suboptimal, leading to ?-cell death and possibly autoimmune triggering. It may be that the pancreatic ?-cell is exceptional in this aspect, because other cell types are known to induce an IFN response when transduced with adenovirus (49, 50).
Previous studies with isolated pancreatic islets have revealed that human ?-cells are clearly more resistant against toxin- and cytokine-induced damage than rodent cells (51, 52). Our present results suggest that this is also the case in adenovirus-induced islet cell death. Moreover, in response to IFN- treatment, human islet cells seem to be more efficient in inducing genes belonging to the antigen processing and antigen presentation MHC class IA family, which might be a critical event in the context of viral transduction.
Genetic engineering of ?-cells for use in gene therapy approaches aims at enhancing the resistance against poor grafting, rejection, and autoimmune attack in type 1 diabetes. For this purpose as well as for basic ?-cell research, viral-derived gene transfer tools should not have any impact by themselves on the function, morphology, or viability of target cells. We showed in this study that prolonged culture of adenoviral-transduced islet cells leads to significant cytotoxicity and that pancreatic islet cells do not have the capacity to induce an IFN response. This process is more evident when high concentrations of the virus are used, and the outcome seems to depend upon the extent of the deletion of the viral genome. If inappropriate viral vectors are chosen in an in vivo situation, pancreatic ?-cells could be directly destroyed by adenovirus-induced cytolysis, or alternatively, the transduction process could cause an inflammatory reaction, leading to or exacerbating the ?-cell-targeted autoimmune response. Considering the wide use of adenoviral vehicles for genetic modification of nondividing cells, these findings should be considered in the design and application of such vectors in experimental and clinical ?-cell research.
Acknowledgments
We gratefully acknowledge the excellent technical assistance of Ing-Marie M?rsare. We also thank Prof. William S. M. Wold (Department of Molecular Microbiology and Immunology, St. Louis University School of Medicine, St. Louis, MO) for providing us with the wild-type adenoviruses.
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