BBRC Biochemical and Biophysical Research Communications 343 (2006) 816–824 www.elsevier.com/locate/ybbrc
Intrasplenic electro-transfer of IL-4 encoding plasmid DNA efficiently inhibits rat experimental allergic encephalomyelitis Seong-Hyun Ho a, Hwang-Jae Lee a, Dong-Sik Kim a, Jae-Gyun Jeong a, Sujeong Kim a, Seung Shin Yu b, Zhe Jin d, Sunyoung Kim a,c, Jong-Mook Kim a,* a
c
ViroMed Co. Ltd., 1510-8 BongCheon-dong, KwanAk-gu, Seoul 151-818, Republic of Korea b TaKaRa Bio Inc., SETA 3-4-1, OTSU, SHIGA 520-2193, Japan Institute of Molecular Biology and Genetics, Seoul National University, Seoul 151-742, Republic of Korea d Zhongshan Hospital of Dlian University, Dalian 116001, People’s Republic of China Received 21 February 2006 Available online 20 March 2006
Abstract Most of the previous studies in which cytokine DNA plasmids were delivered by systemic administration exhibited only marginal therapeutic effects, if any, in the EAE model. One strategy to overcome this limitation would be to determine the optimal delivery route leading to significant beneficial effects both in early (prophylactic) and late (therapeutic) treatments. To address this issue, we directly compared the effects of intrasplenic (IS) and intramuscular (IM) electro-transfer of interleukin-4 (IL-4) DNA in the rat experimental allergic encephalomyelitis (EAE) model. In the preventive experiment, rats received IM (25 or 150 lg) or IS (25 lg) administration of IL-4 DNA followed by in vivo electroporation the day before MBP immunization. In the late treatment experiment, rats were treated with IM (150 lg) or IS (25 lg) administration of IL-4 DNA with electroporation 10 days after MBP immunization. As a control the same amount of vector DNA was used. Macroscopic analysis indicated that the onset of moderate to severe EAE in rats treated with IS transfer of 25 lg of IL-4 DNA was prevented on a significant level compared with IM 25 lg of the IL-4 DNA transfer group or the control group in the preventive experiments. More importantly, IS transfer of 25 lg of IL-4 DNA considerably suppressed the severity of EAE in late treatment experiments while IM transfer of 150 lg of IL-4 DNA had little effect. The MBP-specific expression of IFN-c from stimulated splenocytes was considerably decreased by the IS IL-4 DNA transfer group both in the preventive and therapeutic experiments while IM transfer had this effect only in the preventive protocol. Histological analysis showed that spinal cord inflammation was considerably reduced in the IS IL-4 DNA transfer group. These data provide the first demonstration that IS electro-transfer of IL-4 DNA is more effective both in the prevention and modulation of EAE than IM transfer and that IS electro-gene transfer may present a new approach to cytokine therapy in autoimmune diseases. 2006 Elsevier Inc. All rights reserved. Keywords: Experimental allergic encephalomyelitis; Gene therapy; Interleukin-4; In vivo electroporation
Experimental allergic encephalomyelitis (EAE) is an animal model for human multiple sclerosis (MS) [1]. It is a T-cell mediated demyelinating disease of the central nervous system (CNS) [2]. Pro-inflammatory cytokines (i.e., Th1 cytokines), such as IFN-c, TNF-a, and TNF-b, are believed to play a crucial role in this pathogenic process since they can promote and sustain the development of *
Corresponding author. Fax: +82 2 2102 7280. E-mail address:
[email protected] (J.-M. Kim).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.03.030
myelin-specific T cells and promote the recruitment of peripheral myelinotoxic effector cells (i.e., monocyte/macrophages) [3,4]. Therefore, blockade or regulation of proinflammatory cytokines has been considered as an effective therapeutic strategy for EAE. Apart from direct blockade of IFN-c/TNF by soluble receptor or blocking antibody, regulation can be achieved by modulatory cytokines such as IL-10, TGF-b, and IL-4 [5–8]. Owing to the chronic nature of the disease, gene therapy offers potentially unique advantages over previous protein
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therapy. When compared with various viral and non-viral techniques for gene transfer, naked DNA is probably the safest, simplest, and most inexpensive [9]. Recently, ourselves and others have shown that the gene transfer efficiency of naked DNA could be significantly enhanced by in vivo electroporation, resulting in beneficial effects in the animal model of autoimmune arthritis [10–13]. In this study, we applied this technology for the delivery of IL-4 DNA in the rat EAE model. Most of the previous studies in which cytokine DNA plasmids were delivered by systemic administration exhibited little, if any, therapeutic effects in the EAE model [14–16]. One strategy to overcome this limitation would be to find the optimal delivery route that leads to significant therapeutic effects both in early (prophylactic) and late (therapeutic) treatments. Among the candidate organs to be targeted for electro-transfer of naked DNA, the spleen could be an appealing site. It is one of the most important lymphoid organs, involved in the initiation of immune responses. Because the spleen is enriched in lymphocytes, especially in the area of white pulp, it has been targeted for various immune modulation strategies such as vaccination [17]. Moreover, it has been reported that efficient intrasplenic (IS) gene transfer could be achieved by in vivo electroporation without impairment or over-activation of immune response [18]. The skeletal muscle could be another promising site for electro-gene therapy due to its large size, good capacity for protein synthesis, easy accessibility for intramuscular (IM) injection, and the possibility for repeat injections. Furthermore, the muscle has been shown to cause uptake of DNA and express it for a long time after IM administration [19–21]. In this study, we directly compared the beneficial effects of IS and IM electro-transfer of IL-4 DNA in the rat EAE model. A plasmid DNA encoding IL-4 gene was introduced into the spleens or skeletal muscles of EAE rats, and its effect on EAE was measured by clinical evaluation, histological examination, and in vitro analysis of splenocytes. Materials and methods Plasmid DNA. As a murine IL-4 expression vector, pCK-mIL4, which has been previously described in detail, was used [11]. The plasmids were purified using an EndoFree plasmid Maxi prep kit (Qiagen,Valencia, CA, USA), dissolved in 0.9% NaCl, diluted to 4 lg/ll, and stored at 20 C prior to use. DNA injection and in vivo electroporation. Rats were anesthetized with ketamine (6.75 mg/rat)/xylazine (330 lg/rat). For IM DNA transfer, aliquots of 50 ll or 300 ll of plasmid DNA (pCK-mIL4 or control pCK) at 0.5 lg/ll in 0.9% NaCl were injected into one or two sites in the gastrocnemius muscle of the hind legs (total amount of DNA was 25 or 150 lg per rat). Commercially available two-array needle electrodes (model 530, BTX, San Diego, CA) were used for electroporation. The needle electrodes were applied to the shaved skin on either side of the marked DNA injection point. Consecutively square-wave electrical pulses were administered eight times using an ECM830 pulse generator (BTX, San Diego, CA) at 400 V/cm and a rate of one pulse/s, with each pulse being 20 ms in duration. For IS electro-transfer, after anesthesia, a 2 cm laparatomy was performed on the left flank and the spleen was drawn out from the peritoneal
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cavity. The vasculature of the spleen was clamped and a 30-gauge syringe was then deeply inserted into the spleen and the DNA was injected [18]. A total of 25 lg of plasmid DNA diluted in 50 ll of 0.9% NaCl was used for each rat. Immediately after DNA injection, the caliper electrodes (model 383, BTX) were placed on each side of the spleen, the electric pulses were applied using an ECM830 pulse generator, and the clamp was then removed. The electrical parameters for IS electro-transfer were eight pulses of 20 ms at 200 V/cm at a frequency of 1 Hz. The spleen was then placed back into the peritoneal cavity and the incision was sutured. Induction of EAE and treatment protocol. Lewis rats (Charles River, MA, USA), weighed 200–250 g at the start of experiments, were immunized subcutaneously into the hind footpad with a mixture of purified guinea pig myelin basic protein (MBP) (25 lg/100ll of PBS; Sigma, MO, USA), emulsified in Freund’s complete adjuvant containing Mycobacterium tuberculosis H37Ra (250 lg/100 ll; Sigma) in a final volume of 200 ll. In the preventive experiment, on the day prior to immunization, the rats were divided into six groups and individually treated with either IM electro-transfer of IL-4 or control DNA (25 lg or 150 lg per rat) or IS electro-transfer of IL-4 or control DNA (25 lg per rat). In the late treatment experiment, IM or IS electro-transfer of IL-4 or control DNA (25 lg per rat) was performed 10 days after MBP immunization. The treated rats were monitored for 20 days after immunization and were then sacrificed. Macroscopic scoring of EAE. Clinical EAE scores were evaluated daily for 20 days post-MBP immunization. Clinical signs were scored on a scale from 0 to 4: 0, no clinical signs; 0.5, partial loss of tail tonus; 1, complete loss of tail tonus; 1.5, unsteady gait; 2, paresis of hind legs; 2.5, complete paralysis of the hind legs and/or lower part of the body; 3, paresis of the complete lower part of body up to the diafragma; and 4, death due to EAE [22]. Scoring of clinical EAE grade was carried out by two independent observers who were blinded with regard to the experimental groups. Histopathology. The spinal cords of the rats were removed and fixed in 10% phosphate-buffered formalin for 2 days and then embedded in paraffin. Five micrometer slices of the spinal cord tissue were prepared and stained with hematoxylin and eosin. The tissue was then examined by light microscopy in a blinded manner by a pathologist, evaluated for the extent of inflammation, and then graded as previously described with a slight modification: 0, no inflammation; 1, a few mononuclear cells; 2, organization of inflammatory infiltrates around vessels; 3, extensive peri-vascular cuffing [23]. Measurement of cytokine levels in rat serum or cultured splenocytes. The levels of murine IL-4 in rat sera and the levels of rat IFN-c, rat IL-4, and murine IL-4 in cultured primary spleen cells were measured using commercially available ELISAs for murine IL-4 (Endogen, IL, USA), rat IFN-c (R&D Systems, Minneapolis, MN, USA), and rat IL-4 (R&D Systems), according to the manufacturer’s recommendations. Briefly, the sera were obtained from sacrificed rats and directly subjected to mouse IL4 ELISA without any pretreatment. In the case of rat IFN-c and rat IL-4 levels, splenocytes (1 · 107 cells/ml), which were isolated from IL-4 DNA or control DNA-treated rats, were cultured for 24 h in 24-well plates either with medium containing MBP (10 lmol/ml) or concanavalin A (ConA; 50 lg/ml). Then the supernatants obtained from MBP- or ConA-stimulated splenocytes were directly subjected to ELISA. The MBP-specific induction of IFN-c or IL-4 was calculated by dividing the level of INF-c or IL-4 from MBP-stimulated splenocytes by that from ConA-stimulated splenocytes. For mouse IL-4 levels in supernatant from cultured splenocytes, splenocytes (1 · 107 cells/ml) were isolated from IL-4 DNA or control DNA-treated rats and were cultured for 72 h in 6-well plates, and then supernatant was used for mouse IL-4 ELISA. Real-time quantitative PCR. The presence of transfected pCK-mIL4 DNA in splenocytes or PBMC from the IL-4 DNA or control DNAtreated rats was examined by real-time quantitative PCR using primers specific for pCK-mIL4. The primer sequences were as follows: pCK, 5 0 TCT TTT CTG CAG TCA CC-3 0 ; and mIL4, 5 0 -CTT CTC CTG TGA CCT CGT TC-3 0 . Real-time quantitative PCR was employed using the ABI Prism 7700 sequence detector system (PE Biosystems, Foster City, USA) as previously described [24]. The amount of fluorescence measured in a sample is proportional to the amount of specific PCR product
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generated. CT values corresponding to the cycle number at which the fluorescent emission was monitored in real time reaching a threshold of 10 times the standard deviations of the mean baseline emission from cycle 1 to 15 were measured. Statistical analysis. Differences between experimental groups were tested using the Mann–Whitney rank sum test, unless stated otherwise. P values less than 0.05 were considered significant.
Results Intrasplenic electro-transfer of IL-4 DNA efficiently inhibits EAE in rats As a therapeutic gene for electro-gene therapy of EAE, we chose to use the mouse IL-4 gene because it has demonstrated itself to be functionally active in rats and more importantly the expression of the transferred gene can be easily monitored without interference by the endogenous rat IL-4 protein [25]. As an expression DNA vector, we used pCK, which has been shown to drive high level gene expression in the skeletal and cardiac muscles of mice [11,21,26]. In vivo modulation experiments were performed in the rat EAE model. In the initial series of experiments, we compared the preventive effects of IS and IM electrotransfer of the IL-4 DNA as to the onset and progress of EAE. The day before MBP immunization, rats were divided into six groups. For IM DNA transfer groups, 25 or 150 lg/per rat of IL-4 DNA was injected into one or two selected sites in the skeletal muscles of each rat, followed by in vivo electroporation. As a control, identical amounts of vector DNA lacking the IL-4 coding sequence, pCK, were used for separate groups of rats. In the case of the IS gene transfer group, the spleen of each rat was drawn out from the peritoneal cavity and injected with 25 lg of IL-4 DNA or vector DNA with electroporation. The day following DNA transfer, the mice were immunized with MBP, and then the incidence of EAE was monitored by macroscopic examination every day until 20 days following immunization. As shown in Fig. 1, the incidence of moderate to severe EAE (Pgrade 1.5) was seen in 88% of rats treated with IS electro-transfer of the control DNA, while it was only evident in 46% of rats having received IS transfer of IL-4 DNA (P < 0.05). In contrast, there was no significant difference in the incidence of moderate to severe EAE in 25 lg of the IL-4 DNA group versus the vector DNA group when DNA was transferred by IM administration. A large amount (150 lg) of IL-4 DNA was required to result in statistically significant beneficial effects in the case of IM administration (25% versus 80% in control DNA, P < 0.05). Similarly, the mean accumulated clinical score of EAE was considerably decreased in the rats treated with IS electro-transfer of IL-4 DNA, as compared with that in the control DNA-treated group (P < 0.05) (Fig. 2A). Consistent with the results from incidence analysis, there was no difference in the mean accumulated clinical score between IM 25 lg of the DNA transfer groups (Fig. 2B), while a statistically significant difference was observed
Fig. 1. Effects of electro-transfer of IL-4 DNA on the incidence of EAE in early treatment. On the day before immunization, rats were divided and received an indicated amount of intramuscular (IM) or intrasplenic (IS) electro-transfer of IL-4 DNA (pCK-mIL4) or vector DNA (pCK) (N = 10 per each group). Rats were immunized with guinea pig MBP on day 0. Clinical EAE scores were evaluated on a relative scale 0–4 as described in Materials and methods for 20 days following immunization. The P1.5 score was considered as incidence of moderate to severe EAE. *P < 0.05 versus the control (Fisher’s exact test).
when 150 lg of DNA was transferred by IM injection (Fig. 2C). These results clearly indicated that at the identical dose of IL-4 DNA, IS transfer could more efficiently prevent the progress of EAE than IM transfer. In a second series of experiments, we studied the effect of later treatments. Ten days after MBP immunization, the rats were divided into four groups. For IS DNA transfer groups, 25 lg of IL-4 DNA or control DNA was injected into the spleen of each rat followed by in vivo electroporation. For IM transfer groups, 150 lg of IL-4 DNA or control DNA was transferred to two selected sites of the skeletal muscle of each rat. Macroscopic evaluation on paralysis degree showed that the incidence of moderate to severe EAE (Pgrade 1.5) was significantly decreased in the IS IL-4 DNA transfer group relative to that in the control group (50% versus 100%, P < 0.05) (Fig. 3). In contrast to the preventive experiment, no statistically significant difference in the incidence of moderate to severe EAE was noted between the IM 150 lg of the DNA transfer groups. Twenty days after immunization, the mean accumulated clinical score of the IS IL-4 DNA group was significantly lower than that of the control DNA group (Fig. 4A), while IM transfer of 150 lg of IL-4 DNA had little effect on the mean accumulated clinical score (Fig. 4B). These results demonstrated that in the context of later treatment setting such as treatments 10 days post-immunization, IS DNA transfer, but not IM DNA transfer, could in fact modulate the disease process of EAE. IS electro-transfer of IL-4 DNA reduced the IFN-c expression from MBP-stimulated splenocytes We also assessed the effects of electro-gene transfer on IFN-c expression from MBP-stimulated splenocytes. Splenocytes were obtained from the treated rats that
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Fig. 3. Effects of electro-transfer of IL-4 DNA on the incidence of EAE in late treatment. Rats were immunized with guinea pig MBP on day 0. Ten days after immunization, rats were divided and received an indicated amount of intramuscular (IM) or intrasplenic (IS) electro-transfer of IL-4 DNA (pCK-mIL4) or vector DNA (pCK) (N = 10 per each group). Clinical EAE scores were evaluated on a relative scale 0–4 as described in Materials and methods for 20 days following immunization. The P1.5 score was considered as incidence of moderate to severe EAE. *P < 0.05 versus the control (Fisher’s exact test).
Fig. 2. Effects of electro-transfer of IL-4 DNA on the mean accumulated EAE score in early treatment. On the day before immunization, rats received (A) IS electro-transfer of 25 lg of IL-4 DNA (pCK-mIL4) or vector DNA (pCK), (B) IM electro-transfer of 25 lg of IL-4 DNA or vector DNA, or (C) IM electro-transfer of 150 lg of IL-4 DNA or vector DNA. Clinical EAE scores were evaluated daily on a relative scale 0–4 for 20 days following immunization. Data are means ± SEM of the accumulated EAE score on each day. N = 10 per each group. *P < 0.05 versus the control.
were evaluated in the above experiments (described in Figs. 1–4) on day 20 post-immunization. The expression of IFN-c was examined by incubating splenocytes with MBP (10 lmol/ml) or ConA (50 lg/ml) as described in Materials and methods. The MBP-specific induction of IFN-c was calculated by dividing the level of INF-c from MBP-stimulated splenocytes by that from ConAstimulated splenocytes. When treatment was performed before immunization, the MBP-specific induction of IFN-c was significantly down-regulated both in the rats treated with IM and IS transfer of IL-4 DNA as compared with the control DNA-treated group (P < 0.01) (Figs. 5A and B). However, in the case of late treatment on day 10 post-immunization, IS DNA transfer but not
Fig. 4. Effects of electro-transfer of IL-4 DNA on the mean accumulated EAE score in late treatment. Ten days following immunization, rats received (A) IS electro-transfer of 25 lg of IL-4 DNA (pCK-mIL4) or vector DNA (pCK), or (B) IM electro-transfer of 150 lg of IL-4 DNA or vector DNA. Clinical EAE scores were evaluated daily on a relative scale 0–4 for 20 days following immunization. Data are means ± SEM of the accumulated EAE score on each day. N = 10 per each group. *P < 0.05 versus the control.
IM transfer inhibited the MBP-specific induction of IFN-c from the stimulated splenocytes (P < 0.01) (Figs. 5C and D). We also examined the effects on MBP-specific
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Fig. 5. Effects of electro-transfer of IL-4 DNA on the MBP-specific induction of rat IFN-c from the stimulated splenocytes. Splenocytes were obtained from the rats when the early treatment (A,B) or late treatment (C,D) experiment was terminated. The splenocytes were incubated with MBP or ConA, and the IFN-c expression from the stimulated splenocytes was determined by ELISA. The relative MBP-specific induction was calculated by dividing the value of MBP-specific IFN-c induction of each sample by the mean value of MBP-specific IFN-c induction in the control DNA-treated group. Data are expressed as means ± SEM. *P < 0.01 versus the control.
IL-4 induction from the splenocytes. As shown in Fig. 6, MBP-specific IL-4 induction was up-regulated in the IS IL-4 DNA transfer group irrespective of treatment time point, while IM IL-4 DNA transfer had diverse effects
depending on treatment time point. These results clearly demonstrated that the IS transfer of IL-4 DNA could more consistently modulate the cytokine expression in the EAE model compared with IM transfer.
Fig. 6. Effects of electro-transfer of IL-4 DNA on the MBP-specific induction of rat IL-4 from the stimulated splenocytes. Splenocytes were obtained from the rats when the early treatment (A,B) or late treatment (C,D) experiment was terminated. The splenocytes were incubated with MBP or ConA, and the IL-4 expression from the stimulated splenocytes was determined by ELISA. The relative MBP-specific induction was calculated by dividing the value of MBP-specific IL-4 induction of each sample by the mean value of MBP-specific IL-4 induction in the control DNA-treated group. Data are expressed as means ± SEM. *P < 0.01 versus the control.
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IS electro-transfer of IL-4 DNA reduced the severity of histopathologic changes in EAE rats We also examined the effects of electro-transfer of IL-4 DNA on histopathologic changes in spinal cord of rats that were treated 10 days after immunization. Sections stained with hematoxylin and eosin showed that peri-vascular inflammation was significantly decreased in the spinal cords of rats treated with IS IL-4 DNA transfer (Figs. 7A and B). Consistent with the results from the macroscopic analysis, a statistically significant difference was found in the degree of inflammation between the IS IL-4 DNA transfer group and the control group (P < 0.01) (Fig. 7C). However, there was no considerable difference in the severity of peri-vascular inflammation in the IM IL-4 DNA transfer group compared with the control group (data not shown). Serum level of IL-4 after IM or IS electro-transfer of IL-4 DNA The results of the experiments described above indicated that IS IL-4 DNA transfer with in vivo electroporation could more efficiently modulate the disease process of EAE compared with IM electro-transfer of the same DNA. One simple explanation for this superiority of IS transfer to IM transfer is that the systemic level of IL-4 was more efficiently elevated to a therapeutically sufficient level by IS transfer than IM transfer. To explore such a possibility, we examined the levels of IL-4 in the sera of rats after IM or IS electro-transfer of IL-4 DNA. As shown in Fig. 8, significant levels (higher than 200 pg/ml) of mouse IL-4 (mIL-4) protein were detected in sera for at least 10 days after IM transfer of 150 lg of IL-4 DNA (P < 0.01, compared with control group). In contrast, there were no major differences in serum mIL-4 levels between the IS IL-4 DNA transfer group and the control group. These results indicated that the beneficial effect of IS IL-4 DNA transfer is not achieved by elevation of systemic IL-4 and that an alternative mechanism may be involved. As the next step in elucidation of therapeutic mechanism of IS DNA transfer, we tested whether IL-4 DNA was directly transfected to the splenocytes. Splenocytes were obtained from the rats that received IS IL-4 DNA transfer,
Fig. 8. Serum levels of mouse IL-4 protein over time in rats that received IS or IM electro-transfer of an indicated amount of IL-4 DNA. Data are means ± SEM of mIL-4 measured in five samples. *P < 0.01 versus the control group.
then the presence of IL-4 DNA was evaluated by real-time quantitative PCR. As shown in Fig. 9A, a significant amount of IL-4 DNA was noted in splenocytes from the rats receiving IS IL-4 DNA transfer. We also examined the presence of IL-4 DNA in peripheral blood mononuclear cells (PMBC) from the treated rats. Similar to splenocytes analysis, a considerable amount of IL-4 DNA was detected in PBMC from the rats treated with IS IL-4 DNA transfer. Furthermore, we tested whether the splenocyte from the treated rats could express the transferred mouse IL-4 gene. ELISA on the supernatant from 3-day cultured splenocytes showed that significant levels of mouse IL-4 protein were produced from the cells of rats that received IS DNA transfer compared with the control splenocytes (Fig. 9B). These results indicated that IS electro-transfer of DNA could result in the generation of some splenocytes that contain the transferred DNA and express the encoded protein. Discussion The findings of the present study demonstrated that IS delivery of plasmid DNA containing cDNA for mouse IL-4 by in vivo electroporation could efficiently reduce the incidence and severity of rat EAE both in early and late treatment protocols, compared with IM electro-transfer of the same DNA. It further indicated that IS electro-transfer
Fig. 7. Effects of IS electro-transfer of IL-4 DNA on peri-vascular inflammation in spinal cord in EAE. Hematoxylin–eosin staining of spinal cord tissues from control rats (A) or rats treated with IS transfer of pCK-mIL4 (B). Data are representative of 20 samples. The peri-vascular inflammation could be significantly downgraded in the spinal cords of rats treated with IS IL-4 DNA transfer (C). The score of peri-vascular inflammation was graded as described in Materials and methods. Original magnification: 100·. *P < 0.05 versus the control.
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Fig. 9. (A) Quantitative analysis of IL-4 DNA in splenocytes or PBMC by real-time quantitative PCR. Plasmid DNA was purified from splenocytes (left) or PBMC (right) of rats having received IS electro-transfer of pCK-mIL4 and followed by PCR analysis using specific primers for pCK-mIL4. The relative copy numbers of starting IL-4 DNA were calculated by dividing the IL-4 DNA amount of each sample by the mean amount of IL-4 DNA in control splenocytes or PBMC. (B) Production of mouse IL-4 protein from splenocytes. The culture supernatant from splenocytes of rats that received IS electrotransfer of pCK-mIL4 was analyzed by ELISA. Data are expressed as means ± SEM. *P < 0.01 versus the control.
of IL-4 DNA had beneficial effects without systemic elevation of serum IL-4 levels and resulted in directly transfected splenocytes expressing transferred mouse IL-4 protein. The protective effect of IL-4 in EAE has already been reported [5,27]. IL-4 deficient mice developed a more severe form of EAE [5]. Systemic administration of recombinant IL-4 protein to EAE mice that were generated by adoptive transfer of MBP-specific lymph node cells resulted in amelioration of clinical disease and the induction of MBPspecific Th2 cells [27]. However, in these previous studies, IL-4 was administered systemically before or during initial sensitization, therefore the effects of IL-4 were prophylactic rather than therapeutic. Similarly, systemic administration of IL-4 DNA plasmids has on the whole exhibited only prophylactic effects but little, if any, therapeutic effects [14–16]. Consistent with these previous reports, the IM electro-transfer of IL-4 DNA had beneficial effects only when treatment was performed before immunization in this study. This limitation of previous systemic DNA or protein treatment may be due to the scarce capacity of systemically administered or expressed cytokines to cross the blood– brain barrier (BBB) and accumulate in the CNS where the EAE pathogenic process takes place [28,29]. Indeed, the therapeutic use of Th2 cytokines (IL-10, TGF-b) or use of cytokine agents with an anti-inflammatory profile (IFN-b, TNF-a receptor p55-Ig) in MS patients has so far been generally disappointing [30,31]. In contrast to previous systemic treatment using IL-4 protein of DNA, in this study we showed that intrasplenic electro-transfer of IL-4 DNA could inhibit the disease process of EAE even when treatment was performed 10 days post-immunization. We found that IS IL-4 DNA transfer could (a) reduce the incidence of moderate to severe EAE and the mean accumulated EAE score for 20 days postimmunization; (b) down-regulate the MBP-specific induction of IFN-c expression from splenocytes; (c) up-regulate the MBP-specific induction of IL-4 expression from splenocytes; and (d) reduce the histopathologic findings in spinal cords from the treated rats. We also established that these beneficial effects were achieved without elevation of serum IL-4 levels and that splenocytes that contain IL-4 DNA and express IL-4 protein were generated by IS DNA trans-
fer. One possible mechanism for the beneficial effects of IS DNA transfer may be that the directly transfected splenocytes migrate and play a certain role(s) in the control of disease progression. Tupin et al. [18] already reported that transfected spleen cells by IS DNA transfer could be detected in extrasplenic locations such as auxiliary and mandibulary lymph nodes and thymus. Our observation that a significant amount of IL-4 DNA was present in PBMC from rats treated with IS DNA transfer supports the possibility that directly transfected splenocytes might migrate from spleen to blood. It was already reported that MBP-specific T cells could migrate parathymic lymph nodes to the blood and to the spleen, and then finally to the central nervous system (CNS) in the EAE model [32]. In addition, non-CNS-specific T cells could also be recruited to the inflammatory site of CNS when clinical EAE was present [33]. Furthermore, it was also reported that T-cell hybridoma engineered to express IL-4 moved to CNS and ameliorated EAE without significant elevation of the serum IL-4 level [34]. Similarly ex vivo cloned T cells engineered to express IL-10 have been shown to have disease ameliorating potential [35]. Success of these systemic immunogene therapy strategies using ex vivo engineered T cells supports the hypothesis that directly modified T cells may play an important role in IS electro-gene therapy. When compared with these ex vivo T cell-based therapies, this plasmid DNA transfer method has several advantages [9]. Ex vivo T cell-based therapy requires preparation of patient-specific T cells, followed by in vitro expansion and genetic modification using viral and non-viral vectors. Therefore, this procedure is generally cumbersome, expensive, and time-consuming. In contrast, a large quantity of highly purified plasmid DNA can be readily obtained at a relatively low cost. Furthermore, quality control of DNA production, an important step on an industrial scale, is expected to be much less complicated than other viral and cell-based vectors. To our knowledge, these results provide the first demonstration that intrasplenic electro-transfer of cytokine DNA can efficiently modulate the disease process in an autoimmune disease model and that IS electro-transfer was more effective than IM electro-transfer. Considering the essential
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