- Email: [email protected]
Interleukin-2 gene therapy of chronic neuropathic pain
PII: S 0 3 0 6 - 4 5 2 2 ( 0 2 ) 0 0 0 7 8 - 7
Neuroscience Vol. 112, No. 2, pp. 409^416, 2002 D 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
www.neuroscience-ibro.com
INTERLEUKIN-2 GENE THERAPY OF CHRONIC NEUROPATHIC PAIN M.-Z. YAO,a J.-F. GU,a J.-H. WANG,a L.-Y. SUN,a M.-F. LANG,a J. LIU,a Z.-Q. ZHAOb and X.-Y. LIUa a
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, PR China b
Shanghai Institute of Physiology, Chinese Academy of Sciences, Shanghai 200031, PR China
Abstract2Previous research has revealed an antinociceptive (analgesic) e¡ect of interleukin-2 (IL-2) in central and peripheral nervous systems. Unfortunately IL-2 is very short-lived in vivo, so it is impractical to apply IL-2 for analgesia in clinic. This study was performed to evaluate the e¡ect of intrathecal delivery of human IL-2 gene on rat chronic neuropathic pain induced by chronic constriction injury of the sciatic nerve. Human IL-2 cDNA was cloned into pcDNA3 containing a cytomegalovirus promoter. The paw-withdrawal latency induced by radiant heat was used to measure the pain threshold. The results showed that recombinant human IL-2 had a dose-dependent antinociceptive e¡ect, but that this only lasted for 10^25 min. The pcDNA3-IL-2 or pcDNA3-IL-2/ lipofectamine complex in contrast also showed dose-dependent antinociceptive e¡ects, but these reached a peak at day 2^3 and were maintained for up to 6 days. Liposome-mediated pcDNA3-IL-2 produced a more powerful antinociceptive e¡ect than pcDNA3-IL-2 alone. The paw-withdrawal latencies were not a¡ected by control treatments such as vehicle, lipofectamine, pcDNA3, or pcDNA3-lipofectamine. In the experimental groups, human IL-2 mRNA was detected by reverse transcription-polymerase chain reaction in the lumbar spinal pia mater, dorsal root ganglion, sciatic nerve, and spinal dorsal horn, but not in gastrocnemius muscle. The expressed IL-2 pro¢le detected by western blot coincided with its mRNA pro¢le except it was present in the spinal dorsal horn at a higher level. Furthermore, human IL-2 assayed by enzyme-linked immunosorbent assay in cerebrospinal £uid could still be detected at day 6, but lower than day 3. The antinociceptive e¡ect of pcDNA3-IL-2 could be blocked by naloxone, showing some relationship of the antinociceptive e¡ect produced by IL-2 gene to the opioid receptors. It is hoped that the new delivery approach of a single intrathecal injection of the IL-2 gene described here may be of some practical use as a part of a gene therapy for treating neuropathic pain. D 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: interleukin-2, gene transfer, intrathecal injection, chronic constriction injury, antinociceptive e¡ect.
supported the notion that IL-2 plays an important role in the modulation of neural and neuroendocrine function (Hanisch and Quirion, 1995). Inhibitory functions of IL-2 have been showed in the CNS by decreasing activity in the anterior hypothalamus (Bindoni et al., 1988), suppressing long-term potentiation in the hippocampus and inducing depolarization in hippocampal neurons (Tancredi et al., 1990). We have previously reported that IL-2 had an antinociceptive (analgesic) e¡ect in both central and peripheral nervous systems (Jiang et al., 1994; Wang et al., 1996), inhibited nociceptive responses of the spinal dorsal horn neurons (Zhao et al., 1994), and this antinociceptive e¡ect could be mediated by IL-2 binding to opioid receptors (Wang et al., 1996; Song and Zhao, 2000). The antinociceptive domain is located at the Phe44 , Tyr45 , Tyr107 and Phe117 residues of human IL-2 (hIL-2) (Wang et al., 1997; Jiang et al., 2000a). Microinjection of hIL-2 in intracerebroventricle (i.c.v.), hippocampus or locus coeruleus could increase the pain threshold (Jiang et al., 2000a; Wu et al., 1999; Guo and Zhao, 2000). The antinociceptive e¡ect was related to the increase of Leuenkephalin in paraventricular hypothalamic nucleus and locus coeruleus (Jiang et al., 2000a), the increase of substance P in periaqueductal gray, the decrease of sub-
Interleukin-2 (IL-2), a 15.5-kDa glycoprotein, is not only an important immunoregulatory molecule that enhances T lymphocyte proliferation after antigenic stimulation, but is also an important neuroregulatory molecule in the CNS (Hanisch and Quirion, 1995; Jiang and Lu, 1998). The radioligand binding assay and mRNA detection have shown wide distribution of IL-2 and IL-2 receptor in rat CNS, mainly localized at hippocampus, hypothalamus, cerebellum, neostriatum (Lapchak et al., 1991), and dorsal root ganglion (DRG) neurons (Song et al., 2000). Accordingly, cumulative evidence has strongly
*Corresponding author. Tel.: +86-21-64746127; fax: +86-2164746127. E-mail address: [email protected] (X.-Y. Liu). Abbreviations : ANOVA, analysis of variance; A-O, atlanto-occipital; L-Gal, L-galactosidase ; CCI, chronic constriction injury ; CMV, cytomegalovirus ; CSF, cerebrospinal £uid; DRG, dorsal root ganglion; ELISA, enzyme-linked immunosorbent assay; hIL-2, human interleukin-2; HRP, horseradish peroxidase ; IL-2, interleukin-2 ; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate-bu¡ered saline; PSP, polysaccharide peptide; PWL, paw withdrawal latency ; rIL-2, recombinant hIL-2; RT-PCR, reverse transcription-polymerase chain reaction. 409
NSC 5530 24-5-02
410
M.-Z. Yao et al.
stance P in the spinal cord (Wu et al., 1999) and the reduction of Fos protein in super¢cial dorsal horn (Guo and Zhao, 2000). However, as we have seen, IL-2 has very short half-life in vivo (Lotze et al., 1985; Donohue et al., 1984; Konrad et al., 1990). Systemic administration of IL-2 at pharmacological doses produces short-lived high concentrations of IL-2 in the vasculature and suboptimal levels in the nervous system. It is impractical to apply IL-2 for analgesia in clinic. To achieve curative e¡ect, IL-2 must be continuously administered, resulting in high cost and inconvenience to patients. In order to put the antinociceptive e¡ect of IL-2 into practical use, new delivery approaches have to be explored. Strategies for long-term expression of IL-2 in the local environment might enhance its antinociceptive e¡ect. Gene therapy is one such strategy that can provide sustained local release of IL-2. As yet, two approaches to gene therapy for the management of chronic pain have recently been investigated in animal models of pain, i.e. recombinant adenovirusmediated delivery of antinociceptive molecules to cerebrospinal £uid (CSF) (Finegold et al., 1999) and use of herpes viruses overexpressing antinociceptive peptides (Wilson et al., 1999; Goss et al., 2001), or reducing the expression of endogenous nociceptive molecules in the nervous system (Rydh-Rinder et al., 2001). Both approaches could attenuate or reverse persistent nociceptive states. In this study, we chose a non-viral gene therapy system to evaluate the antinociceptive e¡ect of intrathecal delivery of liposome-mediated hIL-2 gene and ‘naked’ hIL-2 gene on chronic neuropathic pain, and to study its mechanisms of action.
EXPERIMENTAL PROCEDURES
Experimental animals Male Sprague^Dawley rats (240^260 g) were provided by the Shanghai Experimental Animal Center of Chinese Academy of Sciences, and were housed one per cage at a room temperature of 22‡C, with food and water ad libitum and a 12-h light^dark cycle. In animal experiments, all e¡orts were made to minimize both the su¡ering and the number of animals used. All animal experiments were approved by the Administrative Committee of Experimental Animal Care and Use of Shanghai, and conformed to the National Institute of Health guidelines on the ethical use of animals. Chronic constriction injury (CCI) Rats were anesthetized with sodium pentobarbital (40 mg/kg, intraperitoneal, i.p.; supplemented as necessary). The common sciatic nerve was exposed at the mid-thigh level. Four ligatures of 4-O chromic gut were tied loosely around the nerve with about 1 mm spacing between knots. At the time of tying, the ligatures just barely reduce the nerve diameter. Over time, the ligatures evoke intraneural edema, resulting in constriction (Bennett and Xie, 1988). A sham surgery was performed with the sciatic nerve exposed but not ligated. The incision was closed in layers. Upon recovery from anesthesia, the animals were housed post-operatively in individual clear plastic cages with solid £oors covered with 3^6 cm of soft bedding (sawdust), with free access to food and water. The model of CCI was evaluated before lumbar subarachnoid catheterization, and the
rats with the paw-withdrawal latency (PWL) exceeding 6.5 s were eliminated from this study. Lumbar subarachnoid catheterization After CCI for 27 days, rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). A PE-10 catheter (Becton Dickinson, Sparks, MD, USA) was inserted into the lumbar subarachnoid space between lumbar vertebrae 5 (L5) and L6 with the ‘catheter-through-a-needle’ technique (Storkson et al., 1996). The catheter was chronically implanted. The external portion of the chronic indwelling catheter was protected according to Milligan’s method (Milligan et al., 1999). Upon recovery from anesthesia, animals were returned to their home cages. A minimum of 72 h was allowed for recovery before intrathecal injection. Measurement of pain threshold To test pain threshold, paw-withdrawal re£ex was induced by radiant heat. Rats were placed under an inverted clear plexiglass cage (23U18U13 cm) on a piece of 3-mm-thick glass plate. After acclimation, the radiant heat source was positioned under the glass £oor directly beneath the hind paw. The radiant heat source consisted of a high-intensity projection lamp bulb (8 V, 50 W) located 40 mm below the glass £oor and projecting through a 5U10-mm aperture in the top of a movable case. PWL to radiant heat was measured (Hargreaves et al., 1988). A digital timer automatically read the duration between the start of stimuli and the paw withdrawal. The PWL was measured to the nearest 0.1 s. A cuto¡ time of 15 s of irradiation was used to avoid any tissue damage in this study. Construction of expression plasmid The whole-length cDNA of hIL-2 gene was obtained using polymerase chain reaction (PCR) product from the cDNA pool of mononuclear cells from human peripheral blood (Sun et al., 1995). A 462-bp XhoI^EcoRI fragment of the whole-length hIL2 cDNA was cloned into a pcDNA3 empty plasmid (Invitrogen, Carlsbad, CA, USA), which has a cytomegalovirus (CMV) promoter followed by polycloning sites. pcDNA3 or pcDNA3-IL-2 was transformed into Escherichia coli (Strain JM109). Plasmids were prepared by alkaline lysis, puri¢ed by ammonium acetate precipitation with polyethylene glycol extraction. DNA concentration was determined by ultraviolet absorption at 260 nm. Gene transfection and expression assay of hIL-2 in vitro COS-7 cells were grown in Dulbecco’s modi¢ed Eagle’s medium supplemented with 10% fetal bovine serum without antibiotics. The COS-7 cells in 6-well tissue culture plates at about 60% con£uence were co-transfected with 0.5 Wg/well pCMV-L-Gal (Clontech, Palo Alto, CA, USA) and 1 Wg/well pcDNA3 or pcDNA3-IL-2 plasmid respectively mediated by lipofectamine (Life Technologies, Gaithersburg, MD, USA) according to the manufacturer’s protocol. pCMV-L-Gal plasmid, containing the CMV promoter and encoding for L-galactosidase (L-Gal) as a reporter gene, was employed to rectify the transfection e⁄ciency of plasmid. The cell culture supernatant was collected daily for 10 days after transfection. The bioactivity of hIL-2 in culture supernatant was assayed with 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, which utilizing IL-2-dependent murine cell line CTLL-2 (Sladowski et al., 1993). All bioassays were quanti¢ed with IL-2 from Boehringer Mannheim as a standard. The recti¢cation of the transfection e⁄cacy was performed using a chemiluminescent reporter gene assay system to detect L-Gal (Tropix, Bedford, MA, USA). Administration of drugs and delivery of gene For intrathecal injection of recombinant hIL-2 (rIL-2; Hua
NSC 5530 24-5-02
Interleukin-2 gene therapy of chronic neuropathic pain
Xin High Biotechnology, Shanghai, China), after CCI for 30 days, rats were randomized into a control group, i.e. vehicle (10 mM phosphate bu¡er, pH 7.0), and three experimental groups, i.e. 1U104 , 2U104 , and 3U104 IU rIL-2 respectively, n = 12. The total volume of injection was 10 Wl. The PWL was measured 2, 5, 10, 15, 20, 25, 30 min after intrathecal injection. Before injection, animals were lightly anesthetized with ether to avoid struggling and to minimize factitious responses. Pilot studies were practised to ensure that the anesthesia was light enough so as not to alter the PWL 2 min after injection. For intrathecal injection of IL-2 gene, after sham or CCI for 30 days, rats were randomized into ¢ve control groups, i.e. sham, vehicle [5% glucose in phosphate-bu¡ered saline (PBS)], lipofectamine (50 Wg), pcDNA3 (25 Wg), pcDNA3/lipofectamine (25 Wg DNA), and four experimental groups, i.e. pcDNA3-IL-2 (10 Wg), pcDNA3-IL-2/lipofectamine (10 Wg DNA), pcDNA3IL-2 (25 Wg), pcDNA3-IL-2/lipofectamine (25 Wg DNA), n = 12^ 14. 1 Wg of DNA was mixed with 2 Wg of lipofectamine reagent (2 Wg/Wl). The total volume of injection was 30 Wl. The PWL was measured daily for a week after injection. For naloxone block test, after intrathecal plasmid delivery, CCI rats were randomized into four groups, i.e. pcDNA3/lipofectamine, pcDNA3/lipofectamine+naloxone, pcDNA3-IL-2/ lipofectamine and pcDNA3-IL-2/lipofectamine+naloxone, n = 8. 25 Wg of plasmid in a total volume of 30 Wl was injected into subarachnoid space. Naloxone (Sigma, St. Louis, MO, USA) was given (1 mg/kg, i.p.) at day 3 after intrathecal injection of plasmid. The PWL was measured at intervals of 10 min for 90 min after naloxone injection. Reverse transcription-PCR (RT-PCR) for detection of hIL-2 mRNA in di¡erent tissues
411
sected. The freshly dissected tissue samples were homogenized at 4‡C in lysis bu¡er [50 mM Tris^HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 100 Wg/ml phenylmethylsulfonyl £uoride, 1 Wg/ml Aprotinin, 1% Nonidet P-40]. The homogenates were centrifuged at 12 000Ug for 2 min, the supernatants were collected, and stored at 320‡C until hIL-2 western blot and enzyme-linked immunosorbent assay (ELISA). Western blot for detection of hIL-2 in di¡erent tissues and CSF Protein content of tissue crude extracts and CSF after intrathecal injection for 3 days was determined against a standardized control using Bradford colorimetric method (Bradford, 1976). A total of 20 Wg of protein was separated by 12% SDS^PAGE and transferred to nitrocellulose ¢lter paper (Amersham, Buckinghamshire, England). Non-speci¢c binding on the nitrocellulose ¢lter paper was minimized with a blocking bu¡er containing 5% non-fat dry milk in 1UTTBS [25 mM Tris^HCl (pH 7.5), 0.15 M NaCl, 0.05% Tween 20, 0.001% Thimerosal]. The treated ¢lter paper was then incubated, ¢rst with mouse anti-hIL-2 monoclonal antibody (Chemicon, Temecula, CA, USA) or goat anti-rat actin polyclonal antibody serving as loading control (Santa Cruz, CA, USA) and then with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:2000 dilution, Santa Cruz, CA, USA) or HRP-conjugated donkey antigoat IgG (1:5000 dilution, Santa Cruz, CA, USA). The membrane was reacted with a chemiluminescent substrate (Pierce, Rockford, IL, USA) according to the manufacturer’s instruction and the image was obtained by exposing to an X-ray ¢lm. ELISA for hIL-2 level in CSF
After intrathecal injection for 3 days, to obtain tissue for RTPCR, rats were anesthetized with ether, and killed by decapitation. The L4^6 regions of spinal dorsal horn, pia mater, DRG, sciatic nerve and gastrocnemius muscle of rats were freshly dissected. Total tissue RNA was isolated using Trizol reagent (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s protocol. The purity and quantity of the isolated RNA were determined by ultraviolet spectrophotometer (OD260 /OD280 ), after which 2 Wg of total RNA was reversely transcribed into the cDNA. The RNA was incubated at 37‡C for 60 min with a mixture of 200 U of M-MuLV reverse transcriptase (Promega, Madison, WI, USA), 1URT bu¡er, 1.25U1035 mM each of dNTP, 25 U of ribonuclease inhibitor, and 0.2 Wg of DNA random hexamer primer in a volume of 25 Wl. Following this, the reaction mixture was incubated at 95‡C for 5 min to denature the RNA^cDNA hybrid and to inactivate the reverse transcriptase. 1 Wl of RT product was mixed with 2.5 U of Pfu DNA polymerase, 20 pmol each of sense and antisense primers in a bu¡er containing 10 mM of Tris^HCl (pH 8.3), 50 mM of KCl, 2.5 mM of MgCl2 , and 0.25 mM of each dNTP in a volume of 50 Wl. The primers used in this experiment were 5P-ATGTACAGGATGCAACTCCT-3P (sense) and 5P-TCAAGTCAGTGTTGAGATGA-3P (antisense) for hIL-2, and 5P-CCAGCCATGTACGTTGCTATCCAG-3P (sense), 5P-GGAACCGCTCATTGCCAATGGTGA3P (antisense) for L-actin whose expression served as internal control. The ampli¢ed DNA fragments were expected to be 462 bp (hIL-2) and 378 bp (L-actin). The PCR cycles consisted of preincubation at 94‡C for 7 min, denaturation at 94‡C for 1 min, annealing at 40‡C for 10 s, and extension at 72‡C for 30 s, for two cycles, denaturation at 94‡C for 1 min, annealing at 50‡C for 1 min, and extension at 72‡C for 1 min, for 30 cycles, and an extension at 72‡C for 7 min. Collection of CSF and preparation of tissue crude extracts After intrathecal injection for 3 or 6 days, rats were anesthetized with ether, and 10 Wl of CSF was respectively drawn out and stored at 320‡C. Then, the rats were killed by decapitation, and the L4^6 regions of spinal pia mater, dorsal horn, DRG, sciatic nerve, and gastrocnemius muscle were immediately dis-
hIL-2 of CSF was analyzed by ELISA kit (BioSource, Camarillo, CA, USA). Plates were coated with 100 Wl/well mouse antihIL-2 monoclonal antibody at 1.0 Wg/ml and incubated for 18 h at 4‡C, then were blocked by adding 300 Wl of blocking solution to each well and incubated 2 h at room temperature. Standards and CSF were diluted with RPMI containing 10% fetal calf serum, and added at 100 Wl/well followed immediately by addition of 50 Wl/well of biotinylated mouse anti-hIL-2 antibody at 0.4 Wg/ml. The plates were then incubated for 2 h at room temperature with continual shaking. After washes, 100 Wl/well of streptavidin^HRP working solution was added and incubated for 30 min at room temperature. At last, 100 Wl/well of the freshly prepared chromogen tetramethyl benzidine was added and incubated for 30 min at room temperature with continual shaking in the dark followed by addition of 50 Wl/well of stop solution. The optical density of the plates was measured at 450 nm (reference ¢lter 650 nm). Statistical analysis Data were analyzed using Student’s t-test for hIL-2 gene expression assay in vitro, the one-way analysis of variance (ANOVA) for the e¡ect of intrathecal rIL-2 or hIL-2 gene injection on chronic neuropathic pain, an assay of hIL-2 level in CSF after intrathecal gene delivery and the in£uence of naloxone on the antinociceptive e¡ect induced by hIL-2 gene. The post-hoc student Newman^Keuls test was also used for further analysis. Statistical signi¢cance was taken at P 6 0.05. Values were presented as means X S.E.M.
RESULTS
E¡ect of intrathecal administration of rIL-2 on chronic neuropathic pain To evaluate the antinociceptive e¡ect of IL-2 on chronic neuropathic pain, the PWL was measured at various time points from 2 to 30 min after intrathecal
NSC 5530 24-5-02
412
M.-Z. Yao et al.
Fig. 1. Antinociceptive e¡ect of intrathecal injection of rIL-2 on chronic neuropathic pain. After CCI for 30 days, intrathecal injection of vehicle or rIL-2 was performed. rIL-2 (Low), rIL-2 (medium), and rIL-2 (high) were 1U104 , 2U104 and 3U104 IU rIL-2 in a volume of 10 Wl respectively, n = 12. The PWL was measured 2, 5, 10, 15, 20, 25, and 30 min after injection. Baseline is the time point before intrathecal injection. Data are presented as means X S.E.M. Signi¢cance is de¢ned as **P 6 0.01, *P 6 0.05 compared with vehicle injection (ANOVA followed by student Newman^Keuls test).
injection of vehicle and rIL-2. The PWL was not a¡ected by vehicle (P s 0.05, compared with the baseline). rIL-2 (1U104 , 2U104 , 3U104 IU respectively) had dose-dependent antinociceptive e¡ect on neuropathic pain, which reached peak at 5 min after injection. The antinociceptive e¡ect induced by 1U104 , 2U104 , or 3U104 IU rIL-2 respectively lasted 10, 20, or 25 min (P 6 0.05, versus vehicle) (Fig. 1). It demonstrated that intrathecal injection of rIL-2 could alleviate pain with a dose-dependent manner and we therefore could use this delivery way for IL-2 gene therapy of neuropathic pain.
Fig. 2. hIL-2 gene expression assay in vitro. COS-7 cells were cotransfected with pCMV-L-Gal (as an internal control) and pcDNA3 or pcDNA3-IL-2 mediated by lipofectamine. The cell culture supernatant was collected daily for 10 days after transfection. The bioactivity of hIL-2 was assayed with the MTT method, which utilized IL-2-dependent murine cell line CTLL-2. The transfection e⁄ciency was recti¢ed using L-Gal. Data are presented as means X S.E.M., n = 4. Signi¢cance is de¢ned as **P 6 0.01 compared with pcDNA3 transfection at the same day (Student’s t-test).
high dose (25 Wg) of IL-2 gene produced a stronger antinociceptive e¡ect (from day 1 to day 5) than a low dose (10 Wg) (from day 2 to day 5) after intrathecal injection (P 6 0.05, high-dose versus low-dose), which reached peak at day 2 for naked pcDNA3-IL-2 or day 3 for pcDNA3-IL-2/lipofectamine complex. The high-dose pcDNA3-IL-2/lipofectamine complex produced stronger antinociceptive e¡ect than high-dose naked pcDNA3-IL2, so did the low-dose (p 6 0.05, pcDNA3-IL-2/lipofectamine complex versus naked pcDNA3-IL-2) (Fig. 3). It showed that the antinociceptive e¡ect of hIL-2 gene was dose-dependent, and lipofectamine could enhance this
hIL-2 gene expression in vitro To test the expression e⁄ciency of pcDNA3-IL-2 construct, the bioactivity of hIL-2 in COS-7 cells culture supernatant was assayed from day 1 to day 10 after transfection. The transfection e⁄ciency was recti¢ed using L-Gal. There was no hIL-2 production in the culture supernatant after pcDNA3 transfection, whereas the bioactivity of hIL-2 could be detected from day 1 to day 9 after pcDNA3-IL-2 transfection (P 6 0.01, versus pcDNA3 transfection at the same day), which reached peak at day 2 (Fig. 2). The results showed pcDNA3-IL-2 had high expression e⁄ciency. Therapeutic e¡ect of intrathecal hIL-2 gene delivery on chronic neuropathic pain We previously con¢rmed the antinociceptive e¡ect of rIL-2 by intrathecal injection. Here, we employed intrathecal delivery of hIL-2 gene for gene therapy of chronic neuropathic pain. The PWL was measured from day 1 to day 7 after intrathecal injection of indicated reagents. The results showed the PWL was not a¡ected by intrathecal delivery of vehicle, lipofectamine, pcDNA3, or pcDNA3-lipofectamine (P s 0.05, compared with the baseline). Intrathecal delivery of naked pcDNA3-IL-2 or pcDNA3-IL-2/lipofectamine complex showed obviously antinociceptive e¡ect on neuropathic pain, and a
Fig. 3. E¡ect of intrathecal hIL-2 gene delivery on chronic neuropathic pain. After CCI for 30 days, intrathecal injection of 5% glucose in PBS (vehicle, n = 12), 50 Wg of lipofectamine (lipo, n = 12), 25 Wg of pcDNA3 (pcDNA3, n = 13), 25 Wg of pcDNA3+ lipofectamine (pcDNA3/lipo, n = 12), 10 Wg of pcDNA3-IL-2 [pcDNA3-IL-2 (L), n = 14], 10 Wg of pcDNA3-IL-2+lipofectamine [pcDNA3-IL-2/lipo (L), n = 13], 25 Wg of pcDNA3-IL-2 [pcDNA3IL-2 (H), n = 13], 25 Wg of pcDNA3-IL-2+lipofectamine [pcDNA3IL-2/lipo (H), n = 13] respectively, was given. DNA and lipofectamine were mixed with a ratio of 1:2 (Wg:Wg). The total volume of injection was 30 Wl. The PWL was measured daily for a week after injection. Baseline is the time point before intrathecal delivery. Data are presented as means X S.E.M. Signi¢cance is de¢ned as **P 6 0.01, *P 6 0.05 versus pcDNA3/lipo (ANOVA followed by student Newman^Keuls test).
NSC 5530 24-5-02
Interleukin-2 gene therapy of chronic neuropathic pain
Fig. 4. Tissue localization of hIL-2 mRNA expression after intrathecal gene delivery. After 25 Wg of a pcDNA3-IL-2+lipofectamine injection in a volume of 30 Wl for 3 days, hIL-2 mRNA expression was detected by RT-PCR. The L-actin served as a internal control. The DNA fragments of 462 bp (hIL-2) and 378 bp (L-actin) were ampli¢ed. Lane M, marker (pUC mix marker, MBI, Burlington, ON) ; lane 1, spinal dorsal horn; lane 2, spinal pia mater; lane 3, DRG; lane 4, sciatic nerve; lane 5, gastrocnemius muscle; lane C, positive control for hIL-2 plasmid ; lane W, water control.
413
Fig. 5. hIL-2 protein expression in di¡erent tissues and CSF after intrathecal gene delivery. After a 25 Wg of pcDNA3-IL-2+lipofectamine injection in a volume of 30 Wl for 3 days, hIL-2 protein expression was detected by western blot. Actin expression was used as a loading control. The bands of 15.4 kDa (hIL-2) and 43 kDa (Actin) were detected. Lane 1, spinal dorsal horn; lane 2, spinal pia mater; lane 3, DRG ; lane 4, sciatic nerve; lane 5, gastrocnemius muscle; lane 6, CSF.
spinal pia mater could be secreted to the spinal dorsal horn where pain modulation took place. Assay of hIL-2 level in CSF after intrathecal gene delivery
e¡ect. The therapeutic e¡ect of hIL-2 gene could maintain for 5^6 days. Tissue localization of hIL-2 mRNA expression after intrathecal gene delivery To elucidate the molecular mechanism of hIL-2 gene therapy on chronic neuropathic pain, we determined the regions in which hIL-2 mRNA was expressed. The results showed that after 25 Wg of pcDNA3-IL-2/lipofectamine injection for 3 days, when the therapeutic e¡ect of hIL-2 gene was the strongest, hIL-2 mRNA was detected by RT-PCR in regions of the lumbar spinal pia mater, DRG, sciatic nerve, and spinal dorsal horn, but not in gastrocnemius muscle (Fig. 4). The relative expression amount (band’s integrating optical density) of spinal dorsal horn was only 30.21% compared to that of spinal pia mater, that is, the mRNA level in spinal dorsal horn was obviously lower than that in spinal pia mater. It indicated that pcDNA3-IL-2 was principally transfected into spinal pia mater, with only small quantities into spinal dorsal horn. hIL-2 protein expression in di¡erent tissues and CSF after intrathecal gene delivery It is hIL-2 protein that has antinociceptive e¡ect. To elucidate the direct molecular basis of the therapeutic e¡ect of hIL-2 gene in our delivery system, the hIL-2 protein was detected by western blot. hIL-2 protein was found in lumbar spinal pia mater, DRG, sciatic nerve, spinal dorsal horn, and CSF, but not in gastrocnemius muscle. Actin was not detected in CSF. The hIL-2 level in regions of the lumbar spinal pia mater was the highest in all observed tissues, which coincided with the hIL-2 mRNA level in spinal pia mater. However, the relative level of hIL-2 protein of spinal dorsal horn reached 67.25% of spinal pia mater (Fig. 5). Compared with hIL-2 mRNA relative level of spinal dorsal horn, it indicated that the hIL-2 protein expressed in
The amount of hIL-2 protein secreted into CSF could indirectly re£ect the hIL-2 protein level expressed by hIL-2 gene transfected into the tissues mentioned above. To further verify whether the therapeutic e¡ect of hIL-2 gene is related to di¡erent hIL-2 protein levels, ELISA was employed to quantify the hIL-2 level in CSF. The results showed that the hIL-2 expression produced by hIL-2 gene in CSF was also dose-dependent (P 6 0.01, high versus low dose of naked pcDNA3-IL2; P 6 0.01, high versus low dose of pcDNA3-IL-2/lipofectamine complex), and pcDNA3-IL-2/lipofectamine complex produced higher expression than naked pcDNA3-IL-2 (P 6 0.01, both high and low dose of pcDNA3-IL-2/lipofectamine complex versus both high and low dose of naked pcDNA3-IL-2). The hIL-2 expression in CSF at day 6 after pcDNA3-IL-2 delivery could still be detected, but obviously lower than that at day 3 (P 6 0.01, 3 versus 6 days in all IL-2 gene-injected groups). hIL-2 in CSF was not detected by ELISA after vehicle, lipofectamine, pcDNA3 or pcDNA3/lipofectamine were injected (Fig. 6). These results further proved the therapeutic e¡ect of hIL-2 gene was related to di¡erent hIL-2 protein levels. In£uence of naloxone on the antinociceptive e¡ect produced by hIL-2 gene To explore the signaling pathway mechanism of the antinociceptive e¡ect produced by hIL-2 gene, an antagonist of opioid receptor, naloxone, was used at day 3 after intrathecal injection. The PWL was measured at intervals of 10 min for 90 min after naloxone injection. The result showed that PWL in pcDNA3/lipofectamine group was not a¡ected by naloxone (P s 0.05, versus pcDNA3/lipofectamine). However, from 10 min to 50 min after naloxone injection, pcDNA3-IL-2/lipofectamine complex-induced antinociceptive e¡ect could almost be reversed (P 6 0.05, versus pcDNA3-IL-2/lipofectamine) (Fig. 7). It indicated that the opioid receptor might be involved in this antinociceptive process.
NSC 5530 24-5-02
414
M.-Z. Yao et al.
Fig. 6. Assay of hIL-2 level in CSF after intrathecal gene delivery. After intrathecal injection of 5% glucose in PBS (vehicle), 50 Wg of lipofectamine (lipo), 25 Wg of pcDNA3 (pcDNA3), 25 Wg of pcDNA3+lipofectamine (pcDNA3/lipo), 10 Wg of pcDNA3-IL-2 [pcDNA3-IL-2 (L)], 10 Wg of pcDNA3-IL-2+lipofectamine [pcDNA3-IL-2/lipo (L)], 25 Wg of pcDNA3-IL-2 [pcDNA3-IL-2 (H)], 25 Wg of pcDNA3-IL-2+lipofectamine [pcDNA3-IL-2/lipo (H)] in a total volume of 30 Wl for 3 days and 6 days, the level of hIL-2 in CSF was analyzed by ELISA. Sham represents rats performed with the sciatic nerve exposed but not ligated and injected. Data are presented as means X S.E.M. (ng/ml, n = 6). Signi¢cance is de¢ned as **P 6 0.01, compared with pcDNA3/lipo (3 days) ; ## P 6 0.01, compared with 6 days in all IL-2 gene-injected groups. (ANOVA followed by student Newman^Keuls test).
DISCUSSION
Antinociceptive e¡ect of IL-2 The clinical use of IL-2 evoked some idea that IL-2 might have an antinociceptive e¡ect. Animal experiments showed that IL-2 injection (i.c.v.) could apparently increase the pain threshold measured by the tail-£ick method in rats (Jiang et al., 1994), and the experiments in peripheral nervous system gave similar results (Wang et al., 1996). In addition, another experimental evidence of analgesia produced by IL-2 was also found. The Coriolus versicolor polysaccharide peptide (PSP) could relieve acute and chronic pain produced by di¡erent physico-chemical stimulations (Jiang et al., 1991; Teng et al., 1996). This PSP-produced analgesia was blocked by i.c.v. IL-2 antiserum. It indicated that the PSP-produced analgesia was mediated by IL-2 (Gong et al., 1998). In practice, it is a pity that IL-2 showed very short half-life time (t1=2 ) in serum, the ¢rst phase t1=2 approximately 12.9 min, during which most of the exogenous IL-2 was cleared from the serum; and the second phase about 85 min (Konrad et al., 1990). Therefore, it was di⁄cult to apply IL-2 for analgesia in clinic. In order to put the antinociceptive e¡ect of IL-2 into practical use, the IL-2 gene therapy technique was carried out in this study. Choice of vector and gene delivery route The non-viral plasmid vector has advantages of reproducibility and safety. It has been widely used to transfer foreign genes into a variety of mammalian cell types, including both dividing and non-dividing cell types. A few studies have used naked DNA and cationic lipo-
somes to transfer genes into cells of the rodent brain (Schwartz et al., 1996; Brooks et al., 1998). In the brain, as in peripheral tissues, non-viral vectors induce nearly no immune response or toxic e¡ects. In this study, we adopted naked plasmid and liposome-mediated plasmid delivery systems. The route of gene administration is a problem worthy of consideration. The spinal dorsal cord contains the ¢rst set of synaptic contacts that process and relay incoming nociceptive (pain) stimulation. Information related to noxious thermal, mechanical, and chemical stimuli is transmitted from the skin and other peripheral sites to the CNS by myelinated AN and unmyelinated C nociceptors. Both types of neurons synapse in the dorsal horn of spinal cord onto various second-order neurons that either carry information to the thalamus and other brain centers or synapse on other spinal neurons. Injecting drugs into the spinal £uid is a useful tool in the research on analgesia under unanesthetized, unsedated and unrestrained conditions. The method commonly used for catheterization of the lumbar subarachnoid space of rat implies inserting the catheter through the atlanto-occipital (A-O) membrane and moving the catheter caudally along the spinal cord. The method shows the neurological impairment and a considerable morbidity. Therefore, we adopted the method for direct catheterization of the lumbar subarachnoid space. Direct lumbar catheterization has several advantages compared to the A-O method, i.e. decreasing the su¡ering of the animals, the neurological disturbances, the interference with nociceptive functions of the spinal cord and postsurgical deaths (Storkson et al., 1996). However, employing this technique allows the external portion of the chronic indwelling catheters to be easily damaged after surgery. Therefore, we applied an easy and inexpensive method for protecting the external portion of the cathe-
Fig. 7. In£uence of naloxone on the antinociceptive e¡ect produced by IL-2 gene. After CCI for 30 days, intrathecal injection of 25 Wg of pcDNA3+lipofectamine (pcDNA3/lipo) or 25 Wg of pcDNA3-IL-2+lipofectamine (pcDNA3-IL-2/lipo) in a total volume of 30 Wl (n = 8), DNA and lipofectamine were mixed with a ratio of 1:2 (Wg:Wg). Naloxone (1 mg/kg) was given (i.p.) at day 3 after plasmid injection. The PWL was measured at intervals of 10 min for 90 min after naloxone injection. Baseline is the time point before naloxone injection. Data are presented as means X S.E.M. Signi¢cance is de¢ned as **P 6 0.01, *P 6 0.05 compared with pcDNA3-IL-2/lipo injection (ANOVA followed by student Newman^Keuls test).
NSC 5530 24-5-02
Interleukin-2 gene therapy of chronic neuropathic pain
ter that enhanced catheter viability (Milligan et al., 1999). In addition, to judge the e¡ect of intrathecal IL-2 on neuropathic pain, we injected exogenous rIL-2 into the subarachnoid space. The results showed intrathecal rIL-2 had antinociceptive e¡ect on neuropathic pain in this delivery system. Molecular basis of hIL-2 gene-induced antinociceptive e¡ect Meuli-Simmen has reported that spinal cord (intrathecal) injection of either plasmid DNA alone or cationic liposome DNA complexes produces signi¢cant levels of expression of both reporter genes and biologically relevant genes in non-parenchymal cells lining both the brain and the spinal cord (Meuli-Simmen et al., 1999). We observed that the intrathecal delivery of either pcDNA3-IL-2 alone or pcDNA3-IL-2/lipofectamine complex could produce signi¢cant antinociceptive e¡ect on neuropathic pain. To explore the molecular basis of the antinociceptive e¡ect produced by IL-2 gene, we detected and observed the characteristic of hIL-2 expression. hIL-2 mRNA and protein expression were detected in the lumbar spinal pia mater, DRG, sciatic nerve, and spinal dorsal horn, but not in gastrocnemius muscle. The hIL-2 mRNA and protein level in regions of the lumbar spinal pia mater were the highest in all observed tissues coincidently. It indicated that pcDNA3-IL-2 was principally transfected into spinal pia mater. The hIL-2 protein level of spinal dorsal horn was 67.25% of spinal pia mater, whereas the mRNA level in spinal dorsal horn was only 30.21% of spinal pia mater. It indicated that the hIL-2 protein expressed in spinal pia mater could be secreted to the spinal dorsal horn, and the enrichment of hIL-2 protein in spinal cord parenchyma could potentially target secreted hIL-2 within spinal cord regions relevant to pain modulation. These data well explained the molecular basis of IL-2 gene therapy on the neuropathic pain.
415
as an opioid receptor antagonist markedly decreased this e¡ect, indicating the involvement of an opioid receptor in this process. Naloxone was also reported to interfere with other functions of IL-2 (De Sarro et al., 1990). Previous studies have shown that IL-2 and morphine could exert similar e¡ects in various aspects by decreasing intracellular cAMP content, modulating neuroendocrine activity, suppressing a¡erent sensory transmission and serving as Ca2þ channel blockers (Sharma et al., 1975; Plata-Salaman and ¡rench-Mullen, 1993). All the evidence indicated the interplay between IL-2 and opioid receptor. However, there was also report that rIL-2 had marked antinociception on morphine-tolerant rat and rIL-2 antinociception was only partially blocked by naloxone in normal rats. It suggested that there were possibly some other receptors involved in IL-2-induced antinociception process (Song and Zhao, 2000). In this research, we observed the IL-2 gene-induced antinociceptive e¡ect could be reversed by naloxone. Therefore, IL-2 gene-induced antinociception might also be attributed to interaction between IL-2 and the opioid system.
CONCLUSIONS
This is the ¢rst study of directly intrathecal delivery of IL-2 gene for antinociception in order to make it into practical use. These ¢ndings showed the duration of the antinociceptive e¡ect produced by IL-2 gene was much longer than rIL-2 protein, which would have good prospect in practical clinical use. The antinociceptive e¡ect of IL-2 gene was positively correlated with its protein expression level. Unlike analgesic drugs that are administered systemically, intrathecal delivery of a therapeutic pain-killer gene ensures its protein will be secreted in the vicinity of the nerves that conduct pain impulses. Our approach by using a single intrathecal injection of hIL-2 gene expression plasmid to alleviate chronic neuropathic pain is a simple, economical and time-saving method.
Signaling pathway mechanism of IL-2 gene-induced antinociceptive e¡ect The mechanism underlying IL-2-induced peripheral antinociception is incompletely clear so far. Naloxone
Acknowledgements1We wish to thank Gang Pei, Guangcheng Ji, Cheng Huang and Ping Song for their invaluable scienti¢c support.
REFERENCES
Bennett, G.J., Xie, Y.K., 1988. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 33, 87^ 107. Bindoni, M., Perciavalle, V., Berretta, S., Belluardo, N., Diamantstein, T., 1988. Interleukin 2 modi¢es the bioelectric activity of some neurosecretory nuclei in the rat hypothalamus. Brain Res. 462, 10^14. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248^254. Brooks, A.I., Halterman, M.W., Chadwick, C.A., Davidson, B.L., Haak-Frendscho, M., Radel, C., Porter, C., Federo¡, H.J., 1998. Reproducible and e⁄cient murine CNS gene delivery using a microprocessor-controlled injector. J. Neurosci. Methods 80, 137^147. De Sarro, G.B., Masuda, Y., Ascioti, C., Audino, M.G., Nistico, G., 1990. Behavioural and ECoG spectrum changes induced by intracerebral infusion of interferons and interleukin 2 in rats are antagonized by naloxone. Neuropharmacology 29, 167^179. Donohue, J.H., Lotze, M.T., Robb, R.J., Rosenstein, M., Braziel, R.M., Ja¡e, E.S., Rosenberg, S.A., 1984. In vivo administration of puri¢ed Jurkat-derived interleukin-2 in mice. Cancer Res. 44, 1380^1386. Finegold, A.A., Mannes, A.J., Iadarola, M.J., 1999. A paracrine paradigm for in vivo gene therapy in the central nervous system : treatment of chronic pain. Hum. Gene Ther. 10, 1251^1257.
NSC 5530 24-5-02
416
M.-Z. Yao et al.
Gong, S., Zhang, H.Q., Yin, W.P., Yin, Q.Z., Zhang, Y., Gu, Z.L., Qian, Z.M., Tang, P.L., 1998. Involvement of interleukin-2 in analgesia produced by Coriolus versicolor polysaccharide peptides. Acta Pharmacol. Sin. 19, 67^70. Goss, J.R., Mata, M., Goins, W.F., Wu, H.H., Glorioso, J.C., Fink, D.J., 2001. Antinociceptive e¡ect of a genomic herpes simplex virus-based vector expressing human proenkephalin in rat dorsal root ganglion. Gene Ther. 8, 551^556. Guo, H., Zhao, Z.Q., 2000. Inhibition of nociceptive withdrawal re£ex by microinjection of interleukin 2 into rat locus coeruleus. Neuropeptides 34, 216^220. Hanisch, U.K., Quirion, R., 1995. Interleukin-2 as a neuroregulatory cytokine. Brain Res. Rev. 21, 246^284. Hargreaves, K., Dubner, R., Brown, F., Flores, C., Joris, J., 1988. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32, 77^88. Jiang, C.L., Lu, C.L., 1998. Interleukin-2 and its e¡ects in the central nervous system. Biol. Signals Recept. 7, 148^156. Jiang, C.L., Xu, D., Lu, C.L., Wang, Y.X., You, Z.D., Liu, X.Y., 2000a. Interleukin-2: structural and biological relatedness to opioid peptides. Neuroimmunomodulation 8, 20^24. Jiang, C.L., Xu, D., Zheng, Z.C., Fan, P.F., Liu, X.Y., You, Z.D., Song, C.Y., Wang, C.H., Lu, C.L., 1994. The central analgesic e¡ect of IL-2. Chin. J. Appl. Physiol. 10, 322^324. Jiang, C.L., You, Z.D., Lu, C.L., Xu, D., Wang, A.J., Wang, Y.X., Liu, X.Y., 2000b. Leu-enkephalin induced by IL-2 administration mediates analgesic e¡ect of IL-2. NeuroReport 11, 1483^1485. Jiang, J.W., He, L.F., Yang, Q.Y., 1991. Study on analgesic e¡ect of protein bounded polysaccharide of Coriolus versicolor PSP. J. Shanghai Teach. Univ. (Nat. Sci.) 20, 87^89. Konrad, M.W., Hemstreet, G., Hersh, E.M., Mansell, P.W., Mertelsmann, R., Kolitz, J.E., Bradley, E.C., 1990. Pharmacokinetics of recombinant interleukin 2 in humans. Cancer Res. 50, 2009^2017. Lapchak, P.A., Araujo, D.M., Quirion, R., Beaudet, A., 1991. Immunoautoradiographic localization of interleukin 2-like immunoreactivity and interleukin 2 receptors (Tac antigen-like immunoreactivity) in the rat brain. Neuroscience 44, 173^184. Lotze, M.T., Frana, L.W., Sharrow, S.O., Robb, R.J., Rosenberg, S.A., 1985. In vivo administration of puri¢ed human interleukin 2. I. Half-life and immunologic e¡ects of the Jurkat cell line-derived interleukin 2. J. Immunol. 134, 157^166. Meuli-Simmen, C., Liu, Y., Yeo, T.T., Liggitt, D., Tu, G., Yang, T., Meuli, M., Knauer, S., Heath, T.D., Longo, F.M., Debs, R.J., 1999. Gene expression along the cerebral-spinal axis after regional gene delivery. Hum. Gene Ther. 10, 2689^2700. Milligan, E.D., Hinde, J.L., Mehmert, K.K., Maier, S.F., Watkins, L.R., 1999. A method for increasing the viability of the external portion of lumbar catheters placed in the spinal subarachnoid space of rats. J. Neurosci. Methods 90, 81^86. Plata-Salaman, C.R., ¡rench-Mullen, J.M., 1993. Interleukin-2 modulates calcium currents in dissociated hippocampal CA1 neurons. NeuroReport 4, 579^581. Rydh-Rinder, M., Berge, O.G., Hokfelt, T., 2001. Antinociceptive e¡ects after intrathecal administration of phosphodiester-, 2P-O-allyl-, and C-5propyne-modi¢ed antisense oligodeoxynucleotides targeting the NMDAR1 subunit in mouse. Mol. Brain Res. 86, 23^33. Schwartz, B., Benoist, C., Abdallah, B., Rangara, R., Hassan, A., Scherman, D., Demeneix, B.A., 1996. Gene transfer by naked DNA into adult mouse brain. Gene Ther. 3, 405^411. Sharma, S.K., Nirenberg, M., Klee, W.A., 1975. Morphine receptors as regulators of adenylate cyclase activity. Proc. Natl. Acad. Sci. USA 72, 590^594. Sladowski, D., Steer, S.J., Clothier, R.H., Balls, M., 1993. An improved MTT assay. J. Immunol. Methods 157, 203^207. Song, P., Zhao, Z.Q., 2000. Interleukin 2-induced antinociception partially coupled with mu receptor. Cytokine 12, 1240^1242. Song, P., Zhao, Z.Q., Liu, X.Y., 2000. Expression of IL-2 receptor in dorsal root ganglion neurons and peripheral antinociception. NeuroReport 11, 1433^1436. Storkson, R.V., Kjorsvik, A., Tjolsen, A., Hole, K., 1996. Lumbar catheterization of the spinal subarachnoid space in the rat. J. Neurosci. Methods 65, 167^172. Sun, J.T., Qian, G.X., Chen, S.S., Zheng, Z.C., Sun, L.Y., Liu, X.Y., 1995. A study of the construction of di¡erent expression vectors containing IL-2 gene and their expressions in eukaryotic cells. Acta Biochem. Biophys. Sin. 27, 247^252. Tancredi, V., Zona, C., Velotti, F., Eusebi, F., Santoni, A., 1990. Interleukin-2 suppresses established long-term potentiation and inhibits its induction in the rat hippocampus. Brain Res. 525, 149^151. Teng, A.F., Zhang, Y.Y., Gu, Z.L., Yin, Q.Z., Qian, Z.N., Guo, C.Y., Zhou, W.X., 1996. Experimental studies on the analgesic e¡ect of the polysaccharide peptide extracted from Coriolus versicolor. Chin. Pharm. J. 31, 591^594. Wang, Y., Li, L., Liu, J.F., Wang, Z.Y., Jiang, C.L., Liu, X.Y., 1997. The analgesic domain of IL-2. Biochem. Biophys. Res. Commun. 230, 542^ 545. Wang, Y., Pei, G., Cai, Y.C., Zhao, Z.Q., Wang, J.B., Jiang, C.L., Zheng, Z.C., Liu, X.Y., 1996. Human interleukin-2 could bind to opioid receptor and induce corresponding signal transduction. NeuroReport 8, 11^14. Wilson, S.P., Yeomans, D.C., Bender, M.A., Lu, Y., Goins, W.F., Glorioso, J.C., 1999. Antihyperalgesic e¡ects of infection with a preproenkephalin-encoding herpes virus. Proc. Natl. Acad. Sci. USA 96, 3211^3216. Wu, X., Li, H.D., Li, X.C., Ruan, H.Z., Wang, J., 1999. E¡ects of intra-hippocampal injection of interleukin-2 on pain threshold and formaldehyde-induced substance P-like immunoreactivity in periaqueductal gray and spinal cord. Acta Pharmacol. Sin. 20, 839^843. Zhao, Z.Q., Yang, H.Q., Liu, X.Y., 1994. Inhibitory action of immunoregulator interleukin-2 on pain-sensitive spinal neurons in the cat. Chin. J Neurosci. 1, 37^42. (Accepted 21 January 2002)
NSC 5530 24-5-02
Neuroscience Vol. 112, No. 2, pp. 409^416, 2002 D 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
www.neuroscience-ibro.com
INTERLEUKIN-2 GENE THERAPY OF CHRONIC NEUROPATHIC PAIN M.-Z. YAO,a J.-F. GU,a J.-H. WANG,a L.-Y. SUN,a M.-F. LANG,a J. LIU,a Z.-Q. ZHAOb and X.-Y. LIUa a
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, PR China b
Shanghai Institute of Physiology, Chinese Academy of Sciences, Shanghai 200031, PR China
Abstract2Previous research has revealed an antinociceptive (analgesic) e¡ect of interleukin-2 (IL-2) in central and peripheral nervous systems. Unfortunately IL-2 is very short-lived in vivo, so it is impractical to apply IL-2 for analgesia in clinic. This study was performed to evaluate the e¡ect of intrathecal delivery of human IL-2 gene on rat chronic neuropathic pain induced by chronic constriction injury of the sciatic nerve. Human IL-2 cDNA was cloned into pcDNA3 containing a cytomegalovirus promoter. The paw-withdrawal latency induced by radiant heat was used to measure the pain threshold. The results showed that recombinant human IL-2 had a dose-dependent antinociceptive e¡ect, but that this only lasted for 10^25 min. The pcDNA3-IL-2 or pcDNA3-IL-2/ lipofectamine complex in contrast also showed dose-dependent antinociceptive e¡ects, but these reached a peak at day 2^3 and were maintained for up to 6 days. Liposome-mediated pcDNA3-IL-2 produced a more powerful antinociceptive e¡ect than pcDNA3-IL-2 alone. The paw-withdrawal latencies were not a¡ected by control treatments such as vehicle, lipofectamine, pcDNA3, or pcDNA3-lipofectamine. In the experimental groups, human IL-2 mRNA was detected by reverse transcription-polymerase chain reaction in the lumbar spinal pia mater, dorsal root ganglion, sciatic nerve, and spinal dorsal horn, but not in gastrocnemius muscle. The expressed IL-2 pro¢le detected by western blot coincided with its mRNA pro¢le except it was present in the spinal dorsal horn at a higher level. Furthermore, human IL-2 assayed by enzyme-linked immunosorbent assay in cerebrospinal £uid could still be detected at day 6, but lower than day 3. The antinociceptive e¡ect of pcDNA3-IL-2 could be blocked by naloxone, showing some relationship of the antinociceptive e¡ect produced by IL-2 gene to the opioid receptors. It is hoped that the new delivery approach of a single intrathecal injection of the IL-2 gene described here may be of some practical use as a part of a gene therapy for treating neuropathic pain. D 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: interleukin-2, gene transfer, intrathecal injection, chronic constriction injury, antinociceptive e¡ect.
supported the notion that IL-2 plays an important role in the modulation of neural and neuroendocrine function (Hanisch and Quirion, 1995). Inhibitory functions of IL-2 have been showed in the CNS by decreasing activity in the anterior hypothalamus (Bindoni et al., 1988), suppressing long-term potentiation in the hippocampus and inducing depolarization in hippocampal neurons (Tancredi et al., 1990). We have previously reported that IL-2 had an antinociceptive (analgesic) e¡ect in both central and peripheral nervous systems (Jiang et al., 1994; Wang et al., 1996), inhibited nociceptive responses of the spinal dorsal horn neurons (Zhao et al., 1994), and this antinociceptive e¡ect could be mediated by IL-2 binding to opioid receptors (Wang et al., 1996; Song and Zhao, 2000). The antinociceptive domain is located at the Phe44 , Tyr45 , Tyr107 and Phe117 residues of human IL-2 (hIL-2) (Wang et al., 1997; Jiang et al., 2000a). Microinjection of hIL-2 in intracerebroventricle (i.c.v.), hippocampus or locus coeruleus could increase the pain threshold (Jiang et al., 2000a; Wu et al., 1999; Guo and Zhao, 2000). The antinociceptive e¡ect was related to the increase of Leuenkephalin in paraventricular hypothalamic nucleus and locus coeruleus (Jiang et al., 2000a), the increase of substance P in periaqueductal gray, the decrease of sub-
Interleukin-2 (IL-2), a 15.5-kDa glycoprotein, is not only an important immunoregulatory molecule that enhances T lymphocyte proliferation after antigenic stimulation, but is also an important neuroregulatory molecule in the CNS (Hanisch and Quirion, 1995; Jiang and Lu, 1998). The radioligand binding assay and mRNA detection have shown wide distribution of IL-2 and IL-2 receptor in rat CNS, mainly localized at hippocampus, hypothalamus, cerebellum, neostriatum (Lapchak et al., 1991), and dorsal root ganglion (DRG) neurons (Song et al., 2000). Accordingly, cumulative evidence has strongly
*Corresponding author. Tel.: +86-21-64746127; fax: +86-2164746127. E-mail address: [email protected] (X.-Y. Liu). Abbreviations : ANOVA, analysis of variance; A-O, atlanto-occipital; L-Gal, L-galactosidase ; CCI, chronic constriction injury ; CMV, cytomegalovirus ; CSF, cerebrospinal £uid; DRG, dorsal root ganglion; ELISA, enzyme-linked immunosorbent assay; hIL-2, human interleukin-2; HRP, horseradish peroxidase ; IL-2, interleukin-2 ; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate-bu¡ered saline; PSP, polysaccharide peptide; PWL, paw withdrawal latency ; rIL-2, recombinant hIL-2; RT-PCR, reverse transcription-polymerase chain reaction. 409
NSC 5530 24-5-02
410
M.-Z. Yao et al.
stance P in the spinal cord (Wu et al., 1999) and the reduction of Fos protein in super¢cial dorsal horn (Guo and Zhao, 2000). However, as we have seen, IL-2 has very short half-life in vivo (Lotze et al., 1985; Donohue et al., 1984; Konrad et al., 1990). Systemic administration of IL-2 at pharmacological doses produces short-lived high concentrations of IL-2 in the vasculature and suboptimal levels in the nervous system. It is impractical to apply IL-2 for analgesia in clinic. To achieve curative e¡ect, IL-2 must be continuously administered, resulting in high cost and inconvenience to patients. In order to put the antinociceptive e¡ect of IL-2 into practical use, new delivery approaches have to be explored. Strategies for long-term expression of IL-2 in the local environment might enhance its antinociceptive e¡ect. Gene therapy is one such strategy that can provide sustained local release of IL-2. As yet, two approaches to gene therapy for the management of chronic pain have recently been investigated in animal models of pain, i.e. recombinant adenovirusmediated delivery of antinociceptive molecules to cerebrospinal £uid (CSF) (Finegold et al., 1999) and use of herpes viruses overexpressing antinociceptive peptides (Wilson et al., 1999; Goss et al., 2001), or reducing the expression of endogenous nociceptive molecules in the nervous system (Rydh-Rinder et al., 2001). Both approaches could attenuate or reverse persistent nociceptive states. In this study, we chose a non-viral gene therapy system to evaluate the antinociceptive e¡ect of intrathecal delivery of liposome-mediated hIL-2 gene and ‘naked’ hIL-2 gene on chronic neuropathic pain, and to study its mechanisms of action.
EXPERIMENTAL PROCEDURES
Experimental animals Male Sprague^Dawley rats (240^260 g) were provided by the Shanghai Experimental Animal Center of Chinese Academy of Sciences, and were housed one per cage at a room temperature of 22‡C, with food and water ad libitum and a 12-h light^dark cycle. In animal experiments, all e¡orts were made to minimize both the su¡ering and the number of animals used. All animal experiments were approved by the Administrative Committee of Experimental Animal Care and Use of Shanghai, and conformed to the National Institute of Health guidelines on the ethical use of animals. Chronic constriction injury (CCI) Rats were anesthetized with sodium pentobarbital (40 mg/kg, intraperitoneal, i.p.; supplemented as necessary). The common sciatic nerve was exposed at the mid-thigh level. Four ligatures of 4-O chromic gut were tied loosely around the nerve with about 1 mm spacing between knots. At the time of tying, the ligatures just barely reduce the nerve diameter. Over time, the ligatures evoke intraneural edema, resulting in constriction (Bennett and Xie, 1988). A sham surgery was performed with the sciatic nerve exposed but not ligated. The incision was closed in layers. Upon recovery from anesthesia, the animals were housed post-operatively in individual clear plastic cages with solid £oors covered with 3^6 cm of soft bedding (sawdust), with free access to food and water. The model of CCI was evaluated before lumbar subarachnoid catheterization, and the
rats with the paw-withdrawal latency (PWL) exceeding 6.5 s were eliminated from this study. Lumbar subarachnoid catheterization After CCI for 27 days, rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). A PE-10 catheter (Becton Dickinson, Sparks, MD, USA) was inserted into the lumbar subarachnoid space between lumbar vertebrae 5 (L5) and L6 with the ‘catheter-through-a-needle’ technique (Storkson et al., 1996). The catheter was chronically implanted. The external portion of the chronic indwelling catheter was protected according to Milligan’s method (Milligan et al., 1999). Upon recovery from anesthesia, animals were returned to their home cages. A minimum of 72 h was allowed for recovery before intrathecal injection. Measurement of pain threshold To test pain threshold, paw-withdrawal re£ex was induced by radiant heat. Rats were placed under an inverted clear plexiglass cage (23U18U13 cm) on a piece of 3-mm-thick glass plate. After acclimation, the radiant heat source was positioned under the glass £oor directly beneath the hind paw. The radiant heat source consisted of a high-intensity projection lamp bulb (8 V, 50 W) located 40 mm below the glass £oor and projecting through a 5U10-mm aperture in the top of a movable case. PWL to radiant heat was measured (Hargreaves et al., 1988). A digital timer automatically read the duration between the start of stimuli and the paw withdrawal. The PWL was measured to the nearest 0.1 s. A cuto¡ time of 15 s of irradiation was used to avoid any tissue damage in this study. Construction of expression plasmid The whole-length cDNA of hIL-2 gene was obtained using polymerase chain reaction (PCR) product from the cDNA pool of mononuclear cells from human peripheral blood (Sun et al., 1995). A 462-bp XhoI^EcoRI fragment of the whole-length hIL2 cDNA was cloned into a pcDNA3 empty plasmid (Invitrogen, Carlsbad, CA, USA), which has a cytomegalovirus (CMV) promoter followed by polycloning sites. pcDNA3 or pcDNA3-IL-2 was transformed into Escherichia coli (Strain JM109). Plasmids were prepared by alkaline lysis, puri¢ed by ammonium acetate precipitation with polyethylene glycol extraction. DNA concentration was determined by ultraviolet absorption at 260 nm. Gene transfection and expression assay of hIL-2 in vitro COS-7 cells were grown in Dulbecco’s modi¢ed Eagle’s medium supplemented with 10% fetal bovine serum without antibiotics. The COS-7 cells in 6-well tissue culture plates at about 60% con£uence were co-transfected with 0.5 Wg/well pCMV-L-Gal (Clontech, Palo Alto, CA, USA) and 1 Wg/well pcDNA3 or pcDNA3-IL-2 plasmid respectively mediated by lipofectamine (Life Technologies, Gaithersburg, MD, USA) according to the manufacturer’s protocol. pCMV-L-Gal plasmid, containing the CMV promoter and encoding for L-galactosidase (L-Gal) as a reporter gene, was employed to rectify the transfection e⁄ciency of plasmid. The cell culture supernatant was collected daily for 10 days after transfection. The bioactivity of hIL-2 in culture supernatant was assayed with 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, which utilizing IL-2-dependent murine cell line CTLL-2 (Sladowski et al., 1993). All bioassays were quanti¢ed with IL-2 from Boehringer Mannheim as a standard. The recti¢cation of the transfection e⁄cacy was performed using a chemiluminescent reporter gene assay system to detect L-Gal (Tropix, Bedford, MA, USA). Administration of drugs and delivery of gene For intrathecal injection of recombinant hIL-2 (rIL-2; Hua
NSC 5530 24-5-02
Interleukin-2 gene therapy of chronic neuropathic pain
Xin High Biotechnology, Shanghai, China), after CCI for 30 days, rats were randomized into a control group, i.e. vehicle (10 mM phosphate bu¡er, pH 7.0), and three experimental groups, i.e. 1U104 , 2U104 , and 3U104 IU rIL-2 respectively, n = 12. The total volume of injection was 10 Wl. The PWL was measured 2, 5, 10, 15, 20, 25, 30 min after intrathecal injection. Before injection, animals were lightly anesthetized with ether to avoid struggling and to minimize factitious responses. Pilot studies were practised to ensure that the anesthesia was light enough so as not to alter the PWL 2 min after injection. For intrathecal injection of IL-2 gene, after sham or CCI for 30 days, rats were randomized into ¢ve control groups, i.e. sham, vehicle [5% glucose in phosphate-bu¡ered saline (PBS)], lipofectamine (50 Wg), pcDNA3 (25 Wg), pcDNA3/lipofectamine (25 Wg DNA), and four experimental groups, i.e. pcDNA3-IL-2 (10 Wg), pcDNA3-IL-2/lipofectamine (10 Wg DNA), pcDNA3IL-2 (25 Wg), pcDNA3-IL-2/lipofectamine (25 Wg DNA), n = 12^ 14. 1 Wg of DNA was mixed with 2 Wg of lipofectamine reagent (2 Wg/Wl). The total volume of injection was 30 Wl. The PWL was measured daily for a week after injection. For naloxone block test, after intrathecal plasmid delivery, CCI rats were randomized into four groups, i.e. pcDNA3/lipofectamine, pcDNA3/lipofectamine+naloxone, pcDNA3-IL-2/ lipofectamine and pcDNA3-IL-2/lipofectamine+naloxone, n = 8. 25 Wg of plasmid in a total volume of 30 Wl was injected into subarachnoid space. Naloxone (Sigma, St. Louis, MO, USA) was given (1 mg/kg, i.p.) at day 3 after intrathecal injection of plasmid. The PWL was measured at intervals of 10 min for 90 min after naloxone injection. Reverse transcription-PCR (RT-PCR) for detection of hIL-2 mRNA in di¡erent tissues
411
sected. The freshly dissected tissue samples were homogenized at 4‡C in lysis bu¡er [50 mM Tris^HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 100 Wg/ml phenylmethylsulfonyl £uoride, 1 Wg/ml Aprotinin, 1% Nonidet P-40]. The homogenates were centrifuged at 12 000Ug for 2 min, the supernatants were collected, and stored at 320‡C until hIL-2 western blot and enzyme-linked immunosorbent assay (ELISA). Western blot for detection of hIL-2 in di¡erent tissues and CSF Protein content of tissue crude extracts and CSF after intrathecal injection for 3 days was determined against a standardized control using Bradford colorimetric method (Bradford, 1976). A total of 20 Wg of protein was separated by 12% SDS^PAGE and transferred to nitrocellulose ¢lter paper (Amersham, Buckinghamshire, England). Non-speci¢c binding on the nitrocellulose ¢lter paper was minimized with a blocking bu¡er containing 5% non-fat dry milk in 1UTTBS [25 mM Tris^HCl (pH 7.5), 0.15 M NaCl, 0.05% Tween 20, 0.001% Thimerosal]. The treated ¢lter paper was then incubated, ¢rst with mouse anti-hIL-2 monoclonal antibody (Chemicon, Temecula, CA, USA) or goat anti-rat actin polyclonal antibody serving as loading control (Santa Cruz, CA, USA) and then with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:2000 dilution, Santa Cruz, CA, USA) or HRP-conjugated donkey antigoat IgG (1:5000 dilution, Santa Cruz, CA, USA). The membrane was reacted with a chemiluminescent substrate (Pierce, Rockford, IL, USA) according to the manufacturer’s instruction and the image was obtained by exposing to an X-ray ¢lm. ELISA for hIL-2 level in CSF
After intrathecal injection for 3 days, to obtain tissue for RTPCR, rats were anesthetized with ether, and killed by decapitation. The L4^6 regions of spinal dorsal horn, pia mater, DRG, sciatic nerve and gastrocnemius muscle of rats were freshly dissected. Total tissue RNA was isolated using Trizol reagent (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s protocol. The purity and quantity of the isolated RNA were determined by ultraviolet spectrophotometer (OD260 /OD280 ), after which 2 Wg of total RNA was reversely transcribed into the cDNA. The RNA was incubated at 37‡C for 60 min with a mixture of 200 U of M-MuLV reverse transcriptase (Promega, Madison, WI, USA), 1URT bu¡er, 1.25U1035 mM each of dNTP, 25 U of ribonuclease inhibitor, and 0.2 Wg of DNA random hexamer primer in a volume of 25 Wl. Following this, the reaction mixture was incubated at 95‡C for 5 min to denature the RNA^cDNA hybrid and to inactivate the reverse transcriptase. 1 Wl of RT product was mixed with 2.5 U of Pfu DNA polymerase, 20 pmol each of sense and antisense primers in a bu¡er containing 10 mM of Tris^HCl (pH 8.3), 50 mM of KCl, 2.5 mM of MgCl2 , and 0.25 mM of each dNTP in a volume of 50 Wl. The primers used in this experiment were 5P-ATGTACAGGATGCAACTCCT-3P (sense) and 5P-TCAAGTCAGTGTTGAGATGA-3P (antisense) for hIL-2, and 5P-CCAGCCATGTACGTTGCTATCCAG-3P (sense), 5P-GGAACCGCTCATTGCCAATGGTGA3P (antisense) for L-actin whose expression served as internal control. The ampli¢ed DNA fragments were expected to be 462 bp (hIL-2) and 378 bp (L-actin). The PCR cycles consisted of preincubation at 94‡C for 7 min, denaturation at 94‡C for 1 min, annealing at 40‡C for 10 s, and extension at 72‡C for 30 s, for two cycles, denaturation at 94‡C for 1 min, annealing at 50‡C for 1 min, and extension at 72‡C for 1 min, for 30 cycles, and an extension at 72‡C for 7 min. Collection of CSF and preparation of tissue crude extracts After intrathecal injection for 3 or 6 days, rats were anesthetized with ether, and 10 Wl of CSF was respectively drawn out and stored at 320‡C. Then, the rats were killed by decapitation, and the L4^6 regions of spinal pia mater, dorsal horn, DRG, sciatic nerve, and gastrocnemius muscle were immediately dis-
hIL-2 of CSF was analyzed by ELISA kit (BioSource, Camarillo, CA, USA). Plates were coated with 100 Wl/well mouse antihIL-2 monoclonal antibody at 1.0 Wg/ml and incubated for 18 h at 4‡C, then were blocked by adding 300 Wl of blocking solution to each well and incubated 2 h at room temperature. Standards and CSF were diluted with RPMI containing 10% fetal calf serum, and added at 100 Wl/well followed immediately by addition of 50 Wl/well of biotinylated mouse anti-hIL-2 antibody at 0.4 Wg/ml. The plates were then incubated for 2 h at room temperature with continual shaking. After washes, 100 Wl/well of streptavidin^HRP working solution was added and incubated for 30 min at room temperature. At last, 100 Wl/well of the freshly prepared chromogen tetramethyl benzidine was added and incubated for 30 min at room temperature with continual shaking in the dark followed by addition of 50 Wl/well of stop solution. The optical density of the plates was measured at 450 nm (reference ¢lter 650 nm). Statistical analysis Data were analyzed using Student’s t-test for hIL-2 gene expression assay in vitro, the one-way analysis of variance (ANOVA) for the e¡ect of intrathecal rIL-2 or hIL-2 gene injection on chronic neuropathic pain, an assay of hIL-2 level in CSF after intrathecal gene delivery and the in£uence of naloxone on the antinociceptive e¡ect induced by hIL-2 gene. The post-hoc student Newman^Keuls test was also used for further analysis. Statistical signi¢cance was taken at P 6 0.05. Values were presented as means X S.E.M.
RESULTS
E¡ect of intrathecal administration of rIL-2 on chronic neuropathic pain To evaluate the antinociceptive e¡ect of IL-2 on chronic neuropathic pain, the PWL was measured at various time points from 2 to 30 min after intrathecal
NSC 5530 24-5-02
412
M.-Z. Yao et al.
Fig. 1. Antinociceptive e¡ect of intrathecal injection of rIL-2 on chronic neuropathic pain. After CCI for 30 days, intrathecal injection of vehicle or rIL-2 was performed. rIL-2 (Low), rIL-2 (medium), and rIL-2 (high) were 1U104 , 2U104 and 3U104 IU rIL-2 in a volume of 10 Wl respectively, n = 12. The PWL was measured 2, 5, 10, 15, 20, 25, and 30 min after injection. Baseline is the time point before intrathecal injection. Data are presented as means X S.E.M. Signi¢cance is de¢ned as **P 6 0.01, *P 6 0.05 compared with vehicle injection (ANOVA followed by student Newman^Keuls test).
injection of vehicle and rIL-2. The PWL was not a¡ected by vehicle (P s 0.05, compared with the baseline). rIL-2 (1U104 , 2U104 , 3U104 IU respectively) had dose-dependent antinociceptive e¡ect on neuropathic pain, which reached peak at 5 min after injection. The antinociceptive e¡ect induced by 1U104 , 2U104 , or 3U104 IU rIL-2 respectively lasted 10, 20, or 25 min (P 6 0.05, versus vehicle) (Fig. 1). It demonstrated that intrathecal injection of rIL-2 could alleviate pain with a dose-dependent manner and we therefore could use this delivery way for IL-2 gene therapy of neuropathic pain.
Fig. 2. hIL-2 gene expression assay in vitro. COS-7 cells were cotransfected with pCMV-L-Gal (as an internal control) and pcDNA3 or pcDNA3-IL-2 mediated by lipofectamine. The cell culture supernatant was collected daily for 10 days after transfection. The bioactivity of hIL-2 was assayed with the MTT method, which utilized IL-2-dependent murine cell line CTLL-2. The transfection e⁄ciency was recti¢ed using L-Gal. Data are presented as means X S.E.M., n = 4. Signi¢cance is de¢ned as **P 6 0.01 compared with pcDNA3 transfection at the same day (Student’s t-test).
high dose (25 Wg) of IL-2 gene produced a stronger antinociceptive e¡ect (from day 1 to day 5) than a low dose (10 Wg) (from day 2 to day 5) after intrathecal injection (P 6 0.05, high-dose versus low-dose), which reached peak at day 2 for naked pcDNA3-IL-2 or day 3 for pcDNA3-IL-2/lipofectamine complex. The high-dose pcDNA3-IL-2/lipofectamine complex produced stronger antinociceptive e¡ect than high-dose naked pcDNA3-IL2, so did the low-dose (p 6 0.05, pcDNA3-IL-2/lipofectamine complex versus naked pcDNA3-IL-2) (Fig. 3). It showed that the antinociceptive e¡ect of hIL-2 gene was dose-dependent, and lipofectamine could enhance this
hIL-2 gene expression in vitro To test the expression e⁄ciency of pcDNA3-IL-2 construct, the bioactivity of hIL-2 in COS-7 cells culture supernatant was assayed from day 1 to day 10 after transfection. The transfection e⁄ciency was recti¢ed using L-Gal. There was no hIL-2 production in the culture supernatant after pcDNA3 transfection, whereas the bioactivity of hIL-2 could be detected from day 1 to day 9 after pcDNA3-IL-2 transfection (P 6 0.01, versus pcDNA3 transfection at the same day), which reached peak at day 2 (Fig. 2). The results showed pcDNA3-IL-2 had high expression e⁄ciency. Therapeutic e¡ect of intrathecal hIL-2 gene delivery on chronic neuropathic pain We previously con¢rmed the antinociceptive e¡ect of rIL-2 by intrathecal injection. Here, we employed intrathecal delivery of hIL-2 gene for gene therapy of chronic neuropathic pain. The PWL was measured from day 1 to day 7 after intrathecal injection of indicated reagents. The results showed the PWL was not a¡ected by intrathecal delivery of vehicle, lipofectamine, pcDNA3, or pcDNA3-lipofectamine (P s 0.05, compared with the baseline). Intrathecal delivery of naked pcDNA3-IL-2 or pcDNA3-IL-2/lipofectamine complex showed obviously antinociceptive e¡ect on neuropathic pain, and a
Fig. 3. E¡ect of intrathecal hIL-2 gene delivery on chronic neuropathic pain. After CCI for 30 days, intrathecal injection of 5% glucose in PBS (vehicle, n = 12), 50 Wg of lipofectamine (lipo, n = 12), 25 Wg of pcDNA3 (pcDNA3, n = 13), 25 Wg of pcDNA3+ lipofectamine (pcDNA3/lipo, n = 12), 10 Wg of pcDNA3-IL-2 [pcDNA3-IL-2 (L), n = 14], 10 Wg of pcDNA3-IL-2+lipofectamine [pcDNA3-IL-2/lipo (L), n = 13], 25 Wg of pcDNA3-IL-2 [pcDNA3IL-2 (H), n = 13], 25 Wg of pcDNA3-IL-2+lipofectamine [pcDNA3IL-2/lipo (H), n = 13] respectively, was given. DNA and lipofectamine were mixed with a ratio of 1:2 (Wg:Wg). The total volume of injection was 30 Wl. The PWL was measured daily for a week after injection. Baseline is the time point before intrathecal delivery. Data are presented as means X S.E.M. Signi¢cance is de¢ned as **P 6 0.01, *P 6 0.05 versus pcDNA3/lipo (ANOVA followed by student Newman^Keuls test).
NSC 5530 24-5-02
Interleukin-2 gene therapy of chronic neuropathic pain
Fig. 4. Tissue localization of hIL-2 mRNA expression after intrathecal gene delivery. After 25 Wg of a pcDNA3-IL-2+lipofectamine injection in a volume of 30 Wl for 3 days, hIL-2 mRNA expression was detected by RT-PCR. The L-actin served as a internal control. The DNA fragments of 462 bp (hIL-2) and 378 bp (L-actin) were ampli¢ed. Lane M, marker (pUC mix marker, MBI, Burlington, ON) ; lane 1, spinal dorsal horn; lane 2, spinal pia mater; lane 3, DRG; lane 4, sciatic nerve; lane 5, gastrocnemius muscle; lane C, positive control for hIL-2 plasmid ; lane W, water control.
413
Fig. 5. hIL-2 protein expression in di¡erent tissues and CSF after intrathecal gene delivery. After a 25 Wg of pcDNA3-IL-2+lipofectamine injection in a volume of 30 Wl for 3 days, hIL-2 protein expression was detected by western blot. Actin expression was used as a loading control. The bands of 15.4 kDa (hIL-2) and 43 kDa (Actin) were detected. Lane 1, spinal dorsal horn; lane 2, spinal pia mater; lane 3, DRG ; lane 4, sciatic nerve; lane 5, gastrocnemius muscle; lane 6, CSF.
spinal pia mater could be secreted to the spinal dorsal horn where pain modulation took place. Assay of hIL-2 level in CSF after intrathecal gene delivery
e¡ect. The therapeutic e¡ect of hIL-2 gene could maintain for 5^6 days. Tissue localization of hIL-2 mRNA expression after intrathecal gene delivery To elucidate the molecular mechanism of hIL-2 gene therapy on chronic neuropathic pain, we determined the regions in which hIL-2 mRNA was expressed. The results showed that after 25 Wg of pcDNA3-IL-2/lipofectamine injection for 3 days, when the therapeutic e¡ect of hIL-2 gene was the strongest, hIL-2 mRNA was detected by RT-PCR in regions of the lumbar spinal pia mater, DRG, sciatic nerve, and spinal dorsal horn, but not in gastrocnemius muscle (Fig. 4). The relative expression amount (band’s integrating optical density) of spinal dorsal horn was only 30.21% compared to that of spinal pia mater, that is, the mRNA level in spinal dorsal horn was obviously lower than that in spinal pia mater. It indicated that pcDNA3-IL-2 was principally transfected into spinal pia mater, with only small quantities into spinal dorsal horn. hIL-2 protein expression in di¡erent tissues and CSF after intrathecal gene delivery It is hIL-2 protein that has antinociceptive e¡ect. To elucidate the direct molecular basis of the therapeutic e¡ect of hIL-2 gene in our delivery system, the hIL-2 protein was detected by western blot. hIL-2 protein was found in lumbar spinal pia mater, DRG, sciatic nerve, spinal dorsal horn, and CSF, but not in gastrocnemius muscle. Actin was not detected in CSF. The hIL-2 level in regions of the lumbar spinal pia mater was the highest in all observed tissues, which coincided with the hIL-2 mRNA level in spinal pia mater. However, the relative level of hIL-2 protein of spinal dorsal horn reached 67.25% of spinal pia mater (Fig. 5). Compared with hIL-2 mRNA relative level of spinal dorsal horn, it indicated that the hIL-2 protein expressed in
The amount of hIL-2 protein secreted into CSF could indirectly re£ect the hIL-2 protein level expressed by hIL-2 gene transfected into the tissues mentioned above. To further verify whether the therapeutic e¡ect of hIL-2 gene is related to di¡erent hIL-2 protein levels, ELISA was employed to quantify the hIL-2 level in CSF. The results showed that the hIL-2 expression produced by hIL-2 gene in CSF was also dose-dependent (P 6 0.01, high versus low dose of naked pcDNA3-IL2; P 6 0.01, high versus low dose of pcDNA3-IL-2/lipofectamine complex), and pcDNA3-IL-2/lipofectamine complex produced higher expression than naked pcDNA3-IL-2 (P 6 0.01, both high and low dose of pcDNA3-IL-2/lipofectamine complex versus both high and low dose of naked pcDNA3-IL-2). The hIL-2 expression in CSF at day 6 after pcDNA3-IL-2 delivery could still be detected, but obviously lower than that at day 3 (P 6 0.01, 3 versus 6 days in all IL-2 gene-injected groups). hIL-2 in CSF was not detected by ELISA after vehicle, lipofectamine, pcDNA3 or pcDNA3/lipofectamine were injected (Fig. 6). These results further proved the therapeutic e¡ect of hIL-2 gene was related to di¡erent hIL-2 protein levels. In£uence of naloxone on the antinociceptive e¡ect produced by hIL-2 gene To explore the signaling pathway mechanism of the antinociceptive e¡ect produced by hIL-2 gene, an antagonist of opioid receptor, naloxone, was used at day 3 after intrathecal injection. The PWL was measured at intervals of 10 min for 90 min after naloxone injection. The result showed that PWL in pcDNA3/lipofectamine group was not a¡ected by naloxone (P s 0.05, versus pcDNA3/lipofectamine). However, from 10 min to 50 min after naloxone injection, pcDNA3-IL-2/lipofectamine complex-induced antinociceptive e¡ect could almost be reversed (P 6 0.05, versus pcDNA3-IL-2/lipofectamine) (Fig. 7). It indicated that the opioid receptor might be involved in this antinociceptive process.
NSC 5530 24-5-02
414
M.-Z. Yao et al.
Fig. 6. Assay of hIL-2 level in CSF after intrathecal gene delivery. After intrathecal injection of 5% glucose in PBS (vehicle), 50 Wg of lipofectamine (lipo), 25 Wg of pcDNA3 (pcDNA3), 25 Wg of pcDNA3+lipofectamine (pcDNA3/lipo), 10 Wg of pcDNA3-IL-2 [pcDNA3-IL-2 (L)], 10 Wg of pcDNA3-IL-2+lipofectamine [pcDNA3-IL-2/lipo (L)], 25 Wg of pcDNA3-IL-2 [pcDNA3-IL-2 (H)], 25 Wg of pcDNA3-IL-2+lipofectamine [pcDNA3-IL-2/lipo (H)] in a total volume of 30 Wl for 3 days and 6 days, the level of hIL-2 in CSF was analyzed by ELISA. Sham represents rats performed with the sciatic nerve exposed but not ligated and injected. Data are presented as means X S.E.M. (ng/ml, n = 6). Signi¢cance is de¢ned as **P 6 0.01, compared with pcDNA3/lipo (3 days) ; ## P 6 0.01, compared with 6 days in all IL-2 gene-injected groups. (ANOVA followed by student Newman^Keuls test).
DISCUSSION
Antinociceptive e¡ect of IL-2 The clinical use of IL-2 evoked some idea that IL-2 might have an antinociceptive e¡ect. Animal experiments showed that IL-2 injection (i.c.v.) could apparently increase the pain threshold measured by the tail-£ick method in rats (Jiang et al., 1994), and the experiments in peripheral nervous system gave similar results (Wang et al., 1996). In addition, another experimental evidence of analgesia produced by IL-2 was also found. The Coriolus versicolor polysaccharide peptide (PSP) could relieve acute and chronic pain produced by di¡erent physico-chemical stimulations (Jiang et al., 1991; Teng et al., 1996). This PSP-produced analgesia was blocked by i.c.v. IL-2 antiserum. It indicated that the PSP-produced analgesia was mediated by IL-2 (Gong et al., 1998). In practice, it is a pity that IL-2 showed very short half-life time (t1=2 ) in serum, the ¢rst phase t1=2 approximately 12.9 min, during which most of the exogenous IL-2 was cleared from the serum; and the second phase about 85 min (Konrad et al., 1990). Therefore, it was di⁄cult to apply IL-2 for analgesia in clinic. In order to put the antinociceptive e¡ect of IL-2 into practical use, the IL-2 gene therapy technique was carried out in this study. Choice of vector and gene delivery route The non-viral plasmid vector has advantages of reproducibility and safety. It has been widely used to transfer foreign genes into a variety of mammalian cell types, including both dividing and non-dividing cell types. A few studies have used naked DNA and cationic lipo-
somes to transfer genes into cells of the rodent brain (Schwartz et al., 1996; Brooks et al., 1998). In the brain, as in peripheral tissues, non-viral vectors induce nearly no immune response or toxic e¡ects. In this study, we adopted naked plasmid and liposome-mediated plasmid delivery systems. The route of gene administration is a problem worthy of consideration. The spinal dorsal cord contains the ¢rst set of synaptic contacts that process and relay incoming nociceptive (pain) stimulation. Information related to noxious thermal, mechanical, and chemical stimuli is transmitted from the skin and other peripheral sites to the CNS by myelinated AN and unmyelinated C nociceptors. Both types of neurons synapse in the dorsal horn of spinal cord onto various second-order neurons that either carry information to the thalamus and other brain centers or synapse on other spinal neurons. Injecting drugs into the spinal £uid is a useful tool in the research on analgesia under unanesthetized, unsedated and unrestrained conditions. The method commonly used for catheterization of the lumbar subarachnoid space of rat implies inserting the catheter through the atlanto-occipital (A-O) membrane and moving the catheter caudally along the spinal cord. The method shows the neurological impairment and a considerable morbidity. Therefore, we adopted the method for direct catheterization of the lumbar subarachnoid space. Direct lumbar catheterization has several advantages compared to the A-O method, i.e. decreasing the su¡ering of the animals, the neurological disturbances, the interference with nociceptive functions of the spinal cord and postsurgical deaths (Storkson et al., 1996). However, employing this technique allows the external portion of the chronic indwelling catheters to be easily damaged after surgery. Therefore, we applied an easy and inexpensive method for protecting the external portion of the cathe-
Fig. 7. In£uence of naloxone on the antinociceptive e¡ect produced by IL-2 gene. After CCI for 30 days, intrathecal injection of 25 Wg of pcDNA3+lipofectamine (pcDNA3/lipo) or 25 Wg of pcDNA3-IL-2+lipofectamine (pcDNA3-IL-2/lipo) in a total volume of 30 Wl (n = 8), DNA and lipofectamine were mixed with a ratio of 1:2 (Wg:Wg). Naloxone (1 mg/kg) was given (i.p.) at day 3 after plasmid injection. The PWL was measured at intervals of 10 min for 90 min after naloxone injection. Baseline is the time point before naloxone injection. Data are presented as means X S.E.M. Signi¢cance is de¢ned as **P 6 0.01, *P 6 0.05 compared with pcDNA3-IL-2/lipo injection (ANOVA followed by student Newman^Keuls test).
NSC 5530 24-5-02
Interleukin-2 gene therapy of chronic neuropathic pain
ter that enhanced catheter viability (Milligan et al., 1999). In addition, to judge the e¡ect of intrathecal IL-2 on neuropathic pain, we injected exogenous rIL-2 into the subarachnoid space. The results showed intrathecal rIL-2 had antinociceptive e¡ect on neuropathic pain in this delivery system. Molecular basis of hIL-2 gene-induced antinociceptive e¡ect Meuli-Simmen has reported that spinal cord (intrathecal) injection of either plasmid DNA alone or cationic liposome DNA complexes produces signi¢cant levels of expression of both reporter genes and biologically relevant genes in non-parenchymal cells lining both the brain and the spinal cord (Meuli-Simmen et al., 1999). We observed that the intrathecal delivery of either pcDNA3-IL-2 alone or pcDNA3-IL-2/lipofectamine complex could produce signi¢cant antinociceptive e¡ect on neuropathic pain. To explore the molecular basis of the antinociceptive e¡ect produced by IL-2 gene, we detected and observed the characteristic of hIL-2 expression. hIL-2 mRNA and protein expression were detected in the lumbar spinal pia mater, DRG, sciatic nerve, and spinal dorsal horn, but not in gastrocnemius muscle. The hIL-2 mRNA and protein level in regions of the lumbar spinal pia mater were the highest in all observed tissues coincidently. It indicated that pcDNA3-IL-2 was principally transfected into spinal pia mater. The hIL-2 protein level of spinal dorsal horn was 67.25% of spinal pia mater, whereas the mRNA level in spinal dorsal horn was only 30.21% of spinal pia mater. It indicated that the hIL-2 protein expressed in spinal pia mater could be secreted to the spinal dorsal horn, and the enrichment of hIL-2 protein in spinal cord parenchyma could potentially target secreted hIL-2 within spinal cord regions relevant to pain modulation. These data well explained the molecular basis of IL-2 gene therapy on the neuropathic pain.
415
as an opioid receptor antagonist markedly decreased this e¡ect, indicating the involvement of an opioid receptor in this process. Naloxone was also reported to interfere with other functions of IL-2 (De Sarro et al., 1990). Previous studies have shown that IL-2 and morphine could exert similar e¡ects in various aspects by decreasing intracellular cAMP content, modulating neuroendocrine activity, suppressing a¡erent sensory transmission and serving as Ca2þ channel blockers (Sharma et al., 1975; Plata-Salaman and ¡rench-Mullen, 1993). All the evidence indicated the interplay between IL-2 and opioid receptor. However, there was also report that rIL-2 had marked antinociception on morphine-tolerant rat and rIL-2 antinociception was only partially blocked by naloxone in normal rats. It suggested that there were possibly some other receptors involved in IL-2-induced antinociception process (Song and Zhao, 2000). In this research, we observed the IL-2 gene-induced antinociceptive e¡ect could be reversed by naloxone. Therefore, IL-2 gene-induced antinociception might also be attributed to interaction between IL-2 and the opioid system.
CONCLUSIONS
This is the ¢rst study of directly intrathecal delivery of IL-2 gene for antinociception in order to make it into practical use. These ¢ndings showed the duration of the antinociceptive e¡ect produced by IL-2 gene was much longer than rIL-2 protein, which would have good prospect in practical clinical use. The antinociceptive e¡ect of IL-2 gene was positively correlated with its protein expression level. Unlike analgesic drugs that are administered systemically, intrathecal delivery of a therapeutic pain-killer gene ensures its protein will be secreted in the vicinity of the nerves that conduct pain impulses. Our approach by using a single intrathecal injection of hIL-2 gene expression plasmid to alleviate chronic neuropathic pain is a simple, economical and time-saving method.
Signaling pathway mechanism of IL-2 gene-induced antinociceptive e¡ect The mechanism underlying IL-2-induced peripheral antinociception is incompletely clear so far. Naloxone
Acknowledgements1We wish to thank Gang Pei, Guangcheng Ji, Cheng Huang and Ping Song for their invaluable scienti¢c support.
REFERENCES
Bennett, G.J., Xie, Y.K., 1988. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 33, 87^ 107. Bindoni, M., Perciavalle, V., Berretta, S., Belluardo, N., Diamantstein, T., 1988. Interleukin 2 modi¢es the bioelectric activity of some neurosecretory nuclei in the rat hypothalamus. Brain Res. 462, 10^14. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248^254. Brooks, A.I., Halterman, M.W., Chadwick, C.A., Davidson, B.L., Haak-Frendscho, M., Radel, C., Porter, C., Federo¡, H.J., 1998. Reproducible and e⁄cient murine CNS gene delivery using a microprocessor-controlled injector. J. Neurosci. Methods 80, 137^147. De Sarro, G.B., Masuda, Y., Ascioti, C., Audino, M.G., Nistico, G., 1990. Behavioural and ECoG spectrum changes induced by intracerebral infusion of interferons and interleukin 2 in rats are antagonized by naloxone. Neuropharmacology 29, 167^179. Donohue, J.H., Lotze, M.T., Robb, R.J., Rosenstein, M., Braziel, R.M., Ja¡e, E.S., Rosenberg, S.A., 1984. In vivo administration of puri¢ed Jurkat-derived interleukin-2 in mice. Cancer Res. 44, 1380^1386. Finegold, A.A., Mannes, A.J., Iadarola, M.J., 1999. A paracrine paradigm for in vivo gene therapy in the central nervous system : treatment of chronic pain. Hum. Gene Ther. 10, 1251^1257.
NSC 5530 24-5-02
416
M.-Z. Yao et al.
Gong, S., Zhang, H.Q., Yin, W.P., Yin, Q.Z., Zhang, Y., Gu, Z.L., Qian, Z.M., Tang, P.L., 1998. Involvement of interleukin-2 in analgesia produced by Coriolus versicolor polysaccharide peptides. Acta Pharmacol. Sin. 19, 67^70. Goss, J.R., Mata, M., Goins, W.F., Wu, H.H., Glorioso, J.C., Fink, D.J., 2001. Antinociceptive e¡ect of a genomic herpes simplex virus-based vector expressing human proenkephalin in rat dorsal root ganglion. Gene Ther. 8, 551^556. Guo, H., Zhao, Z.Q., 2000. Inhibition of nociceptive withdrawal re£ex by microinjection of interleukin 2 into rat locus coeruleus. Neuropeptides 34, 216^220. Hanisch, U.K., Quirion, R., 1995. Interleukin-2 as a neuroregulatory cytokine. Brain Res. Rev. 21, 246^284. Hargreaves, K., Dubner, R., Brown, F., Flores, C., Joris, J., 1988. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32, 77^88. Jiang, C.L., Lu, C.L., 1998. Interleukin-2 and its e¡ects in the central nervous system. Biol. Signals Recept. 7, 148^156. Jiang, C.L., Xu, D., Lu, C.L., Wang, Y.X., You, Z.D., Liu, X.Y., 2000a. Interleukin-2: structural and biological relatedness to opioid peptides. Neuroimmunomodulation 8, 20^24. Jiang, C.L., Xu, D., Zheng, Z.C., Fan, P.F., Liu, X.Y., You, Z.D., Song, C.Y., Wang, C.H., Lu, C.L., 1994. The central analgesic e¡ect of IL-2. Chin. J. Appl. Physiol. 10, 322^324. Jiang, C.L., You, Z.D., Lu, C.L., Xu, D., Wang, A.J., Wang, Y.X., Liu, X.Y., 2000b. Leu-enkephalin induced by IL-2 administration mediates analgesic e¡ect of IL-2. NeuroReport 11, 1483^1485. Jiang, J.W., He, L.F., Yang, Q.Y., 1991. Study on analgesic e¡ect of protein bounded polysaccharide of Coriolus versicolor PSP. J. Shanghai Teach. Univ. (Nat. Sci.) 20, 87^89. Konrad, M.W., Hemstreet, G., Hersh, E.M., Mansell, P.W., Mertelsmann, R., Kolitz, J.E., Bradley, E.C., 1990. Pharmacokinetics of recombinant interleukin 2 in humans. Cancer Res. 50, 2009^2017. Lapchak, P.A., Araujo, D.M., Quirion, R., Beaudet, A., 1991. Immunoautoradiographic localization of interleukin 2-like immunoreactivity and interleukin 2 receptors (Tac antigen-like immunoreactivity) in the rat brain. Neuroscience 44, 173^184. Lotze, M.T., Frana, L.W., Sharrow, S.O., Robb, R.J., Rosenberg, S.A., 1985. In vivo administration of puri¢ed human interleukin 2. I. Half-life and immunologic e¡ects of the Jurkat cell line-derived interleukin 2. J. Immunol. 134, 157^166. Meuli-Simmen, C., Liu, Y., Yeo, T.T., Liggitt, D., Tu, G., Yang, T., Meuli, M., Knauer, S., Heath, T.D., Longo, F.M., Debs, R.J., 1999. Gene expression along the cerebral-spinal axis after regional gene delivery. Hum. Gene Ther. 10, 2689^2700. Milligan, E.D., Hinde, J.L., Mehmert, K.K., Maier, S.F., Watkins, L.R., 1999. A method for increasing the viability of the external portion of lumbar catheters placed in the spinal subarachnoid space of rats. J. Neurosci. Methods 90, 81^86. Plata-Salaman, C.R., ¡rench-Mullen, J.M., 1993. Interleukin-2 modulates calcium currents in dissociated hippocampal CA1 neurons. NeuroReport 4, 579^581. Rydh-Rinder, M., Berge, O.G., Hokfelt, T., 2001. Antinociceptive e¡ects after intrathecal administration of phosphodiester-, 2P-O-allyl-, and C-5propyne-modi¢ed antisense oligodeoxynucleotides targeting the NMDAR1 subunit in mouse. Mol. Brain Res. 86, 23^33. Schwartz, B., Benoist, C., Abdallah, B., Rangara, R., Hassan, A., Scherman, D., Demeneix, B.A., 1996. Gene transfer by naked DNA into adult mouse brain. Gene Ther. 3, 405^411. Sharma, S.K., Nirenberg, M., Klee, W.A., 1975. Morphine receptors as regulators of adenylate cyclase activity. Proc. Natl. Acad. Sci. USA 72, 590^594. Sladowski, D., Steer, S.J., Clothier, R.H., Balls, M., 1993. An improved MTT assay. J. Immunol. Methods 157, 203^207. Song, P., Zhao, Z.Q., 2000. Interleukin 2-induced antinociception partially coupled with mu receptor. Cytokine 12, 1240^1242. Song, P., Zhao, Z.Q., Liu, X.Y., 2000. Expression of IL-2 receptor in dorsal root ganglion neurons and peripheral antinociception. NeuroReport 11, 1433^1436. Storkson, R.V., Kjorsvik, A., Tjolsen, A., Hole, K., 1996. Lumbar catheterization of the spinal subarachnoid space in the rat. J. Neurosci. Methods 65, 167^172. Sun, J.T., Qian, G.X., Chen, S.S., Zheng, Z.C., Sun, L.Y., Liu, X.Y., 1995. A study of the construction of di¡erent expression vectors containing IL-2 gene and their expressions in eukaryotic cells. Acta Biochem. Biophys. Sin. 27, 247^252. Tancredi, V., Zona, C., Velotti, F., Eusebi, F., Santoni, A., 1990. Interleukin-2 suppresses established long-term potentiation and inhibits its induction in the rat hippocampus. Brain Res. 525, 149^151. Teng, A.F., Zhang, Y.Y., Gu, Z.L., Yin, Q.Z., Qian, Z.N., Guo, C.Y., Zhou, W.X., 1996. Experimental studies on the analgesic e¡ect of the polysaccharide peptide extracted from Coriolus versicolor. Chin. Pharm. J. 31, 591^594. Wang, Y., Li, L., Liu, J.F., Wang, Z.Y., Jiang, C.L., Liu, X.Y., 1997. The analgesic domain of IL-2. Biochem. Biophys. Res. Commun. 230, 542^ 545. Wang, Y., Pei, G., Cai, Y.C., Zhao, Z.Q., Wang, J.B., Jiang, C.L., Zheng, Z.C., Liu, X.Y., 1996. Human interleukin-2 could bind to opioid receptor and induce corresponding signal transduction. NeuroReport 8, 11^14. Wilson, S.P., Yeomans, D.C., Bender, M.A., Lu, Y., Goins, W.F., Glorioso, J.C., 1999. Antihyperalgesic e¡ects of infection with a preproenkephalin-encoding herpes virus. Proc. Natl. Acad. Sci. USA 96, 3211^3216. Wu, X., Li, H.D., Li, X.C., Ruan, H.Z., Wang, J., 1999. E¡ects of intra-hippocampal injection of interleukin-2 on pain threshold and formaldehyde-induced substance P-like immunoreactivity in periaqueductal gray and spinal cord. Acta Pharmacol. Sin. 20, 839^843. Zhao, Z.Q., Yang, H.Q., Liu, X.Y., 1994. Inhibitory action of immunoregulator interleukin-2 on pain-sensitive spinal neurons in the cat. Chin. J Neurosci. 1, 37^42. (Accepted 21 January 2002)
NSC 5530 24-5-02