Fever of recombinant human interferon-alpha is

Journal of Neuroimmunology 156 (2004) 107 – 112 www.elsevier.com/locate/jneuroim

Fever of recombinant human interferon-alpha is mediated by opioid domain interaction with opioid receptor inducing prostaglandin E2 Yun-Xia Wanga,b, Wei-Gang Xua, Xue-Jun Suna, Yi-Zhang Chenb, Xin-Yuan Liuc, Hao Tangd, Chun-Lei Jianga,* a

Department of Nautical Medicine, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, P.R. China b Department of Neurobiology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, P.R. China c Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Science, Shanghai 200031, P.R. China d Department of Physiology, China Medical University, Shenyang 110001, P.R. China Received 29 March 2004; received in revised form 22 July 2004; accepted 23 July 2004

Abstract We have reported that there are distinct domains in Interferon-alpha (IFNa) molecule mediating immune and opioid-like effects respectively. And the opioid effect of IFNa is mediated by A opioid receptor. We report here the structural basis of fever induced by recombinant human IFNa. Two kinds of IFNa mutants were obtained and used to investigate the structural basis of fever of IFNa, which are 129Ser-IFNa and 38Leu-IFNa. The antiviral activity of 129Ser-IFNa almost disappeared, but there still retained the strong analgesic activity. The antiviral activity of 38Leu-IFNa remained, but the analgesic activity disappeared completely. It showed that IFNa and 129Ser-IFNa decreased cAMP production, induced the fever, and stimulated PGE2 to release from the hypothalamus slices, which could be blocked by naloxone, but 38Leu-IFNa failed. It is the first demonstration that fever induced by IFNa is mediated by opioid domain of IFNa interacting with opioid receptor. It is inferred that high-activity and low side-effect IFNa or other cytokines could be obtained after being changed the motifs in the tertiary structure. D 2004 Elsevier B.V. All rights reserved. Keywords: Structure basis; cAMP; Pyrogen; Opioid domains; PGE2

1. Introduction Fever induced by administration of Interferon-alpha (IFNa) has been well documented. The structural bases that IFNa interacts with some receptors mediating fever in tertiary structure are not well understood, although several studies indicate that the fever induced by IFNa is related to opioid receptors. Intravenous or intracerebroventricular injection of IFNa into rabbits, cats and mice produces fever that does not involve endotoxin (Dinarello et al., 1984). IFNa could decrease the neuronal activity of

* Corresponding author. Tel.: +86 21 65492382; fax: +86 21 65492382. E-mail address: [email protected] (C.-L. Jiang). 0165-5728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2004.07.013

warm sensitive neurons, while increase the activity of cold sensitive neurons in the preoptic/anterior hypothalamus (PO/AH) area in an opioid-receptor-dependent way. The actions of IFNa on the thermosensitive neurons were blocked by the opioid receptor antagonist naloxone, and not by sodium salicylate in doses which effectively blocked the neuronal response to endotoxin and leukocytic pyrogen (Nakashima et al., 1995, 1998; Hori et al., 1998). We ever reported that there were distinct domains in IFNa molecule that mediate immune and opioid-like effects, respectively, and inferred that the analgesic domain locates around the 122nd Tyr residue of IFNa molecule in tertiary structure (Wang et al., 2000), and the opioid effect of IFNa is mediated by A opioid receptor (Jiang et al., 2000a).

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Y.-X. Wang et al. / Journal of Neuroimmunology 156 (2004) 107–112

2. Materials and methods 2.1. Generation of human IFNa mutants The procedure of mutagenesis of IFNa gene used was the method described by Horton (1997). The mutated cDNA was then sequenced, and the desired mutant cDNA was cloned into the expression vector. 2.2. Purification of wild type and mutant IFNa proteins The purification of IFNa proteins was directed by Hua Xin High Biotech, where the human recombinant IFNa for clinical usage is produced. Fermentation of Escherichia coli DH5a harboring normal or various mutated IFNa genes was performed in M9 medium at 37 8C for 8 h. The bacteria were harvested by centrifugation and then suspended in phosphates buffer solution and sonicated. IFNa and its analog protein were purified by affinity chromatography on an antiIFNa antibody sepharose column (Anhui Anke High Biotechology, China). 2.3. Antiviral activity assay of IFNa The antiviral activity of IFNa was determined in a cytopathic effect reduction assay using human WISH cells challenged with vesicular stomatitis virus (VSV) (Van Heuvel et al., 1986). 2.4. Animals and surgery Adult male Sprague-Dawley (SD) rats (160–180 g) maintained on a 12:12 h light/dark cycle were stereotaxically implanted with stainless steel guide cannulae, under pentobarbital anaesthesia (35 mg/kg, i.p.), to permit intracerebroventricular infusion (i.c.v.) (Jiang et al., 2000b). After surgery, a minimum of 72 h was allowed for recovery before experiments were carried out. Some 8–12 SD rats were used in every group. 2.5. Application of drugs A total of 8 pmol IFNa and its mutants were injected into the lateral ventricle in a volume of 5 Al. 200 pmol Naloxone hydrochloride (NLX, Sigma) was dissolved in warm (37 8C) sterile saline. The vehicle (0.9% saline) was used as control.

square current (mA) (every 1 min) at the moment of response as PT. The range of the current intensity is from 0.2 to 1.2 mA. The intensity of current was measured at 5 and 10 min after IFNa injection and at every 10 min. The percentage of the PT changes was calculated on the basis of mean value of three PT before. 2.7. cAMP assay CHO cells transfected with A opioid receptor (Prof. Pei G., Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Science) were maintained in DMEM (Gibco-BRL) supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2. Some 4105 cells were seeded into each well of a 12-well plate and grew for 24 h. After removal of culture medium and a brief wash with PBS, the cells in presence of 0.5 mM IBMX (1-methyl-3isobutylxanthine, Sigma) were challenged respectively with PBS, forskolin (10 AM Sigma), and forskolin plus IFNa (1 ng/ml) at 37 8C for 15 min. The reaction was terminated with 1 M perchloric acid and neutralized with 2 M K2CO3. After centrifugation at 12,000g for 10 min, aliquot of the supernatants was taken to measure the cAMP levels by 125 I-cAMP kits. The percentages of cAMP production were calculated as [cAMP ( f o r s k o l i n + I F N a) cAMP ( b a s a l ) ]/ [cAMP(forskolin) cAMPB(basal)]100%. 2.8. Pyrogen assay Rectal temperature (Tre) was measured using a thermometer inserted 8 cm into the rectum and held in place by adhesive tape wrapped around the base of the tail (Zawada et al., 1997). The Tre was monitored and recorded at 10 min intervals in a room maintained at 24F1 8C. The Tre in each animal was allowed to stabilize for at least 90 min before any injections were made. All drug solutions were prepared in pyrogen-free glassware and containers. All solutions were sterile, nonpyrogenic and, as an added precaution, were passed through 0.22-Am Millipore bacterial filters. Rats were allowed free water, but deprived of food for 18 h to avoid temperature changes due to food ingestion. The rats were kept at room temperature in plastic restrainers to prevent excessive motion. Only rats which showed stable body temperature in the range of 36.5–37.5 8C for at least 90 min were used for the fever experiments. 2.9. Hypothalamic incubations and radioimmunoassay

2.6. Analgesia assay The electro-stimulation-induced tail-flick was used to measure the pain threshold (PT) of rats according to the method described before (Yang and Lin, 1992; Jiang et al., 2000a). The two electrodes were located subcutaneous at 3 cm from tail tip of the rat. The distance of two electrodes was 1 cm. We took the mean value of three intensities of the

5-day-old Sprague-Dawley pups were decapitated and their brains were quickly isolated. Three hundred micrometre-thick slices of hypothalamic tissue were cut using tissue chopper. The PO/AH explants were placed on millicell-centimeter filter inserts (Millipore, pore size 0.4 Am diameter 30 mm). Three-explants were placed on a single filter inserts. Slice culture media consisted of


Dulbecco’s modified Eagle’s medium (DMEM 75%, GIBCO), heated-inactivated horse serum (25%, GIBCO), and 100 U/ml penicillin and 80 U/ml streptomycin. After 3day culture, medium was replaced with a serum-free medium (B27 supplied with 1 mM glutamin, GIBCO). The medium was changed every 3 days. On the tenth day of culture, hypothalamic sections were incubated with morphine (10 AM), naloxone (20 AM) and IFNa (1 ng/ml), respectively, for 1 h. Then the supernatants were collected after centrifugation at 100 g, and immediately frozen, stored at 70 8C until assayed (Maurer and Wray, 1997; Scott et al., 1987). Prostaglandin E2 (PGE2) was assayed using radioimmunoassay kit with a specific anti-PGE2 antibody (Beijing Biotinge-Tech). All the data are here expressed by meanFSD. Statistical analysis was conducted with variance test.

3. Results

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Fig. 1. Analgesic effects of IFNa and its mutants in CNS. The changes of PT at 20 min after IFNa and its mutants injection with 8 pmol into the lateral ventricle of the rats were shown. IFNa could increase the PT of rats. The mutant 38Leu-IFNa (38L-IFNa) lost analgesic activity completely. However, 129Ser-IFNa (129S-IFNa) still maintained a strong analgesic activity (**pb0.01 vs. control).

3.1. Antiviral activity of IFNa and its mutants By using site-directed mutagenesis, the 38th Phe of IFNa (IFNa2b throughout this experiment) was mutated to Ser, Ala and Leu, respectively. IFNa and its analog proteins were obtained by expression and purification (the purity was more than 95%). And then the antiviral activity of IFNa and its mutants (38Ser-IFNa, 38Ala-IFNa and 38Leu-IFNa) was determined. Table 1 showed that after the 38th Phe residue of human IFNa was mutated to Ser or Ala, the antiviral activity of the mutants (38Ser-IFNa or 38Ala-IFNa) almost disappeared. However, the antiviral activity of 38Leu-IFNa still maintained 71% compared with wild type of IFNa. After the 129th Tyr residue of human IFNa was mutated to Ser, the antiviral activity almost disappeared.

after IFNa injection with 8 pmol. The analgesic effects were shown significantly at 5 min and reached the maximum at 20 min after IFNa injection. However, the analgesic activity of 38Leu-IFNa disappeared completely compared with wild type of IFNa. After the 129th Tyr residue of human IFNa was mutated to Ser, there still maintained the strong analgesic activity Fig. 1 showed the analgesic activity at 20 min after IFNa or its mutants injection. 3.3. Effects of IFNa and its mutants on cAMP content

3.2. Analgesic effect of IFNa and its mutants

Table 2 showed the effects of IFNa and its mutants on cAMP content in CHO cells transfected with A-opioid receptor, and the influence of naloxone on these effects. We could find that IFNa and 129Ser-IFNa could decrease cAMP content, and the effect could be blocked by

IFNa was of significant analgesic action in CNS. The PT was increased significantly compared with the control

Table 2 Influence of IFNa and its mutants on cAMP content in CHO-A cells

Table 1

Group

cAMP content (%)

Antiviral activity of IFNa and its mutants

Control Morphine Morphine+NLX IFNa IFNa+NLX 129Ser-IFNa 129Ser-IFNa+NLX 38Leu-IFNa

100 55.32* 86.26# 83.30* 90.90# 68.16* 83.58# 108.18

Mutation

Antiviral activity (106 U/mg)

Percentage of wild type (%)

Wild type of IFNa Phe38YSer Phe38YAla Phe38YLeu Tyr129YSer

56.00 b0.01 b0.01 40.10 2.69

100 – – 71.6 4.8

The mutants 38Ser-IFNa and 38Ala-IFNa lost antiviral activity completely. However, the mutant 38Leu-IFNa still maintained 71% activity compared with wild type of IFNa. After the 129th Tyr residue of human IFNa was mutated to Ser, the antiviral activity almost disappeared (8 pmol IFNa and its mutants were injected).

IFNa or 129Ser-IFNa (1 ng/ml) could decrease the cAMP content. Naloxone (NLX, 20 AM) could block the effect. 38Leu-IFNa could even raise the cAMP production, but there was no significant difference. Morphine (10 AM) was used as positive control. * pb0.05 vs. control. # pb0.05 vs. morphine or IFNa or 129S-IFNa.

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3.5. Stimulation of rat hypothalamic PGE2 production by IFNa and its mutants Rat hypothalamic slices were incubated with IFNa or its mutants. As shown in Fig. 3, both IFNa and 129Ser-IFNa could stimulate PGE2 production, and these effects were blocked by pretreated with naloxone. 38Leu-IFNa had no significant effect on PGE2 production. Fig. 2. Changes in rectal temperature of rats at 120 min after IFNa injection. IFNa or 129Ser-IFNa (129S-IFNa) injecting with 8 pmol into the lateral ventricle could increase Tre, which could be blocked by naloxone (NLX, 20AM), pretreated. 38Leu-IFNa (38L-IFNa) failed to increase T re of rats (**pb0.01 vs. control; ##pb0.01 vs. IFNa or 129S-IFNa).

naloxone. However, 38Leu-IFNa failed to decrease cAMP content. 3.4. Changes in rectal temperature of rats after IFNa injection The effects of IFNa and its mutants on rectal temperature (Tre) of rats, and the influence of naloxone on these effects were shown in Fig. 2. No difference in basal values of Tre was observed in rats treated with saline. The temperature increased significantly in groups injected with IFNa or 129Ser-IFNa whose antiviral activity almost disappeared, but it still retained the strong opioid-like analgesic activity. However, after the 38th Phe residue of human IFNa was mutated to Leu, the effect of increasing Tre of the rats almost disappeared. In the groups pretreated with naloxone before IFNa or 129Ser-IFNa injection, the change of Tre was not significant compared with saline.

4. Discussion IFNas are a group of molecules synthesized and secreted by macrophage, monocytes, T lymphocytes. They have several biological properties including antiviral activity, antitumor and enhancement of immune function, so they have been widely used in clinical trials. Fever has been the most consistent side effect of IFNa therapy. IFNa, derived from blood leukocytes or cell lines, and recombinant human IFNa have produced fever in nearly all recipients during clinical trials (Dunnick and Galasso, 1980). In this experiment, we provided further information about the pyrogenic action of IFNa. Dinarello et al. (1984) reported that fever of IFNa is not due to endotoxin, but is induced by PGE2 production in the hypothalamus. IFNa could alter the activity of thermosensitive neurons in the PO/AH area, which was blocked by the opioid receptor antagonist naloxone, suggesting that the opioid receptor mechanism is involved in the fever induced by IFNa (Nakashima et al., 1987, 1995, 1998). Actually, IFNa binds to opioid receptors (Blalock and Smith, 1981; Smith and Blalock, 1981; Menzies et al., 1992). We also provided further information that the opioidlike analgesic effect of IFNa is mediated by the A-opioid

Fig. 3. PGE2 production in rat hypothalamic tissue. PGE2 production increased significantly after rat hypothalamic slices were incubated with 1 ng/ml IFNa or 129Ser-IFNa (129S-IFNa), which could be blocked by naloxone (NLX, 20AM). 38Leu-IFNa (38L-IFNa) failed to increase PGE2 production (**pb0.01 vs. control; ##pb0.01 vs. IFNa or 129S-IFNa).


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receptor (Jiang et al., 2000a; Wang et al., 2001). In this experiment, both Tre rise and PGE2 production induced by IFNa could be blocked by naloxone, suggesting an opioid receptor-dependent way. Both structural and functional similarities have been demonstrated between IFNa and endorphins (Blalock and Smith, 1980, 1981; Jiang et al., 2000c). Also, there are significant cross reactivities between IFNa and anti-opioid sera, suggesting the strong antigenic relatedness between IFNa molecule and opioid peptides. We have reported that there are distinct domains in IFNa molecule that mediate immune and opioid-like effects respectively, and inferred that the analgesic domain locates around the 122nd Tyr residue of IFNa molecule in tertiary structure (Wang et al., 2000). A common characteristic of endogenous opioid peptides (EOPs) is the Tyr-X-X-Phe sequence in the amino termini, among which the aromatic residues, Tyr and Phe, are crucial for the analgesic activity of EOPs. And the appropriate spacer between these two aromatic residues is necessary for opipid receptor binding and producing bioactivity, because opioid receptor recognition requires specific three-dimensional structures of the ligand molecules with the correct array of three pharmacophoric groups. Fig. 4 shows that the 38th Phe is near the opioid domain around the 122nd Tyr in the tertiary structure of IFNa, though the distance between the two residues is rather long in the primary structure. A three-dimensional Tyr X X Phe motif in the IFNa that is shared with endogenous opiates, so that IFNa can interfere with the opiate receptor and then induce the fever. So in this study, we changed the 38th Phe residue around the 122nd Tyr residue of IFNa molecule in tertiary structure. After the

38th Phe residue was mutated to Leu, the analgesic activity disappeared completely, although the antiviral activity still maintained 71% compared with wild type of IFNa. We inferred that the 38th Phe is crucial for the opioid-like analgesic effect of IFNa and is one of the constituents of the analgesic domain of IFNa. The opioid-like domain of IFNa consists of the 122nd Tyr and the residues around the 122nd in the tertiary structure, such as the 38th Phe residue. During this study, two kinds of IFNa mutants were used to investigate the structural basis of fever of IFNa, which are 129Ser-IFNa and 38Leu-IFNa. The antiviral activity of 129Ser-IFNa almost disappeared, but there still retained the strong analgesic activity. However, the antiviral activity of 38Leu-IFNa remained, but the analgesic activity disappeared completely. We could find that IFNa and 129Ser-IFNa decreased cAMP production, which could be blocked by naloxone, but 38Leu-IFNa failed to decrease cAMP content. These results indicate that there are different receptor mechanisms inducing immune and opioid-like neuroregulatory effects of IFNa, and inferred that the opioid-like domain of IFNa could bind to opioid receptor to induce intracellular signal transduction. IFNa or 129Ser-IFNa whose antiviral activity almost disappeared could induce the fever, and this effect could be blocked by naloxone, and 38Leu-IFNa had no this effect. It is suggested that the fever of IFNa be mediated by opioidlike domain interacting with opioid receptor. Furthermore, IFNa and 129Ser-IFNa could stimulate PGE2 release from the hypothalamus slices, suggesting that IFNa is pyrogenic by inducing PGE2 in the hypothalamus. And the effect of PGE2 production induced by IFNa and 129Ser-IFNa could be blocked by naloxone. The IFNa mutant, 38Leu-IFNa, failed to stimulate the PGE2 production, suggesting that the opioid domain of IFNa and opioid receptor mechanism mediate the fever of IFNa. Fever induced by IFNa is mediated by opioid domain of IFNa interacting with opioid receptor. It is inferred that highactivity and low side-effect IFNa or other cytokines could be obtained after changing the motifs in the tertiary structure. We have proposed that the multiple domains of cytokines are one of the structural bases of the immunoregulatory and neuroregulatory effects of cytokines. A possible mechanism of actions of cytokines on the immune system and/or CNS exists: multiple actions of cytokines may be mediated by distinct domains or by functional sites of cytokines interacting with different receptors or receptor subtypes (Jiang et al., 1998). The results obtained from this experiment are consistent with the opinion proposed, and provide further information about neuroregulatory effects of cytokines.

Fig. 4. Computer graphics of protein conformations of the tertiary structure of IFNa (MMDB Id: 6520; PDB Id: IRH2). The opioid-like domain of IFNa consists of the 122nd Tyr and the residues around the 122nd in the tertiary structure, which include the 38th Phe residues. The distance between the 122nd Tyr and the 38th Phe residues is 5.856 2.

Acknowledgements This work was supported by National 973 Program of China (G1999054003), National Natural Sciences Founda-

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tion of China (30070710, 30300122) and Shanghai-Unilever Research and Development Fund (2009).

References Blalock, J.E., Smith, E.M., 1980. Human leukocyte interferon: Structural and biological relatedness to adrenocorticotropic hormone and endorphins. Proc. Natl. Acad. Sci. U. S. A. 77, 5972 – 5974. Blalock, J.E., Smith, E.M., 1981. Human leukocyte interferon (HuIFN-a): potent endorphin-like opioid activity. Biochem. Biophys. Res. Commun. 101, 472 – 478. Dinarello, C.A., Bernheim, H.A., Duff, G.W., Le, H.V., Nagabhushan, T.L., Hamilton, N.C., Coceani, F., 1984. Mechanisms of fever induced by recombinant human interferon. J. Clin. Invest. 74, 906 – 913. Dunnick, J.K., Galasso, G.J., 1980. Update on clinical trials with exogenous interferon. J. Infect. Dis. 142, 293 – 299. Hori, T., Katafuchi, T., Take, S., Shimizu, N., 1998. Neuroimmunomodulatory actions of hypothalamic interferon-a. Neuroimmunomodulation 5, 172 – 177. Horton, R.M., 1997. In vitro recombination and mutagenesis of DNA. In: White, B.A. (Ed.), Methods in Molecular Biology: PCR Cloning Protocols, vol. 67. The Humana Press, Totowa, NJ, pp. 141 – 149. Jiang, C.L., Lu, C.L., Liu, X.Y., 1998. The molecular basis for bidirectional communication between the immune and neuroendocrine systems. Domest. Anim. Endocrinol. 15, 363 – 369. Jiang, C.L., Song, L.X., Lu, C.L., You, Z.D., Wang, Y.X., Sun, L.Y., Cui, R.Y., Liu, X.Y., 2000a. Analgesic effect of interferon-alpha via mu opioid receptor in the rats. Neurochem. Int. 36, 193 – 196. 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 effect of IL-2. NeuroReport 11, 1483 – 1485. Jiang, C.L., Xu, D., Lu, C.L., Wang, Y.X., You, Z.D., Liu, X.Y., 2000. Interleukin-2: structural and biological relatedness to opioid peptides. Neuroimmunomodulation 8, 20 – 24.

Maurer, J.A., Wray, S., 1997. Luteinizing hormone-releasing hormone (LHRH) neurons maintained in hypothalamic slice explant cultures exhibit a rapid LHRH mRNA turnover rate. J. Neurosci. 17, 9481 – 9491. Menzies, R.A., Patel, R., Hall, N.R.S., OTGrady, M.P., Rier, S.E., 1992. Human recombinant interferon alpha inhibits naloxone binding to rat brain membranes. Life Sci. 50, PL227 – PL232. Nakashima, T., Hori, T., Kuriyama, K., Kiyohara, T., 1987. Naloxone blocks the interferon-a induced changes in hypothalamic neuronal activity. Neurosci. Lett. 82, 332 – 336. Nakashima, T., Murakami, T., Murai, Y., Hori, T., Miyota, S., Kiyohara, T., 1995. Naloxone suppresses the rising phase of fever induced by interferon-a. Brain Res. Bull. 37, 61 – 66. Nakashima, T., Hori, T., Kuriyama, K., Matsuda, T., 1998. Effects of interferon-a on the activity of preoptic thermosensitive neurons in tissue slices. Brain Res. 454, 361 – 367. Scott, I.M., Fertel, R.H., Boulant, J.A., 1987. Leukocytic pyrogen effects on prostaglandins in hypothalamic tissue slices. Am. J. Physiol. 253, R71 – R76. Smith, E.M., Blalock, J.E., 1981. Human lymphocyte production of corticotrophin and endorphin-like substances: association with leukocyte interferon. Proc. Natl. Acad. Sci. U. S. A. 78 (12), 7530 – 7534. Van Heuvel, M., Bosveld, I.J., Mooren, A.T.A., Trapman, J., Zwarthoff, E.C., 1986. Properties of natural and hybrid murine alpha interferon. J. Gen. Virol. 67, 2215 – 2222. Wang, Y.X., Jiang, C.L., Lu, C.L., Song, L.X., You, Z.D., Shao, X.Y., Cui, R.Y., Liu, X.Y., 2000. Distinct domains of IFNa mediate immune and analgesic effects respectively. J. Neuroimmunol. 108, 64 – 67. Wang, Y.X., Cui, G.Y., Shen, J., Huang, A.J., Liu, X.Y., Chen, Y.Z., Jiang, C.L., 2001. Analgesic domains of interferon-a. NeuroReport 12, 857 – 859. Yang, J., Lin, B.C., 1992. Hypothalamic paraventricular nucleus plays a role in acupuncture analgesia through the central nervous system in the rat. Acupunct. Electro-Ther. Res. 17, 209 – 220. Zawada, W.M., Clarke, J., Ruwe, W.D., 1997. Naloxone differentially alters fevers induced by cytokines. Neurochem. Int. 30, 441 – 448.