Induction of nociceptive responses by intrathecal injection of interleukin-1 in mice

Life SC~QWS,Vol. 65, No. 3, pp. 255-261. 1999 lZOp)TigbtOl999Elrcviasciaaosh. FtintediahUSA. Allrighbreaavcd ELSEVIER

PII SOO24-3205@9)00244-1

INDUCTION OF NOCICEPTIVE RESPONSES BY INTRATHECAL INJECTION OF INTERLEUKIN-1 IN MICE’

Takeshi Tadano”‘, Miyuki Namioka”, Osamu Nakagawasaia, Koichi Tan-No”, Kouji Matsushimab, Yasuo Endo”, and Kensuke Kisaraa “Department of Pharmacology, Tohoku College of Pharmacy, 4-4-l Komatsushima, Aoba-ku, Sendai, 981 Japan; bDepartment of Molecular Preventive Medicine, Division of Social Medicine, Graduate School of Medicine, Tokyo University, 7-3-l Hongo, Bunkyo-ku, Tokyo 113, Japan; “Department of Pharmacology, School of Dentistry, Tohoku University, 4-l Seiryo-machi, Aoba-ku, Sendai, 980 Japan (I&eked in final formMarch 22, 1999) Summary Intrathecal (i.t.) injection (between lumbar vertebrae 5 and 6) into mice of a markedly low dose of ILla (3~10~ fmol or 5.4 fg in 5 p1 per mouse) induced behaviors involving scratching, biting, and licking of non-stimulated hindpaws. The IL-linduced behaviors appeared within 10 min of the injection of IL-la, peaked at 20-40 min, and had disappeared 60 min after the injection. The ILl-induced behaviors were similar to the nociceptive responses induced in mice by i. t. injection of substance P (SP) or subcutaneous (s.c.) injection of formalin into the footpad. The IL-l-induced behaviors were suppressed by intraperitoneal morphine, indicating that they are nociceptive responses. The nociceptive responses induced by 3x104(5.4 fg) of IL-la were almost completely suppressed by co-injection of 0.3 fmol(7.2 pg) of an IL-1 receptor antagonist (ILlra). An antiserum against substance P, but not an antiserum against somatostatin, suppressed the IL-l-induced nociceptive responses. The nociceptive responses induced by S.C. injection of 2 % formalin into the footpad were also inhibited by i. t. injection of 30 pmol (720 ng) of IL-lra. These results suggest that IL1 may play a role in hyperalgesia in mice by acting as a factor augmenting pain transmission in the spinal cord at least in part by either directly or indirectly releasing substance P . Key Words: intedeukin-1 , substance P, pi& spinal cord, aeeieqtive behavior Recent studies have shown that interleukin-1 (IL-l) is a modulator of pain. Intracerebroventricular injections of IL1 induce hyperalgesia (1,2) or analgesia (1,3). Which effect is obtained depends on its dosage: higher doses (ng order/mouse or rat) of IL1 induce analgesia (1,3), while lower doses (pg order/rat) induce hyperalgesia (2). On the other hand, peripheral injections of IL1 mostly induce hyperalgesia (4-7), and stimulation of polymodal receptors by IL1 has been suggested to lead to hyperalgesia (7). Studies on the spinal cord pathway for the transmission of the peripheral sense ‘The results of this work were largely reported at the 70th Annual Meeting of the Japanese pharmacological Society, March 1997. Jpn J Pharmacol73 (Supplement I) 174P (1997). Address for Correspondence: Takeshi Tadano, Department of Pharmacology, Tohoku College of Pharmacy, 4-4-l Komatsushima, Aoba-ku, Sendai, 981 Japan. Fax: +81-22-275-2013

256

Nociceptive Response to Inbatheeal IL-1

Vol. 65, No. 3 1999

of pain to the brain include, for example, experiments in which the subcutaneous (SC.) injection of for&in into the footpad induced the release of neurotransmitters such as substance P (sp), somatostatm (SOM), and glutamate in the spinal cord of rats (8,9). Earlier, Hylden and Wilcox (10) had found that intrathecal (i.t.) injection of SP itself into mice induces a caudally-directed biting and scratching behaviors. However, there are only a few reports concerning the relationship between IL-1 and pain in the spinal cord (11,12), and there are no reports at all on this topic in the mouse. In the present study on mice, we found that i. t. injection of IL1 itself induces nociceptic behaviors similar to those induced by SP, and that similar nociceptic behaviors induced by the S.C. injection of formalin into the footpad can be inhibited by i.t. injection of a recombinant IL-l-receptor antagonist (IL-lra). Materials and Methods Animals : Male ddY mice (Shizuoka Laboratory Center, Shizuoka, Japan) weighing 20-22 g were used in all experiments. The animals were housed under controlled conditions with regard to temperature (22 2 2 “C), humidity (55 + 5 %), and the light-dark cycle (12 h). They were provided with food and water ad libitum until the day of the experiment. All experiments conformed to national requirements (Japanese law no. 105, notification no .6) and complied with the Guidelines for Care and Use of Laboratory Animals in Tohoku College of Pharmacy. Reagents: Recombinant human interleukin-lo (IL-laXmolecular weight 18,000) was provided by Dainippon Pharmaceutical Co. (Osaka, Japan). A recombinant mouse IL1 receptor antagonist (ILlra) (molecular weight about 24,000) was provided by Professor K. Matsushima (Tokyo University)(l3). Morphine hydrochloride (Sankyo, Tokyo, Japan), an antiserum against substance P (SP) (Genosys Biotechnologies, Inc. England), an antiserum against somatostatin (SOM)(Medac Gesellschaft fur Klinishe Spezialpraparate mbh, Germany), and formalin (Kant0 Chemical Co. Tokyo, Japan) were obtained commercially. All drugs were dissolved in sterile saline. Intrathecaf injections: Intrathecal (i.t.) injection into unane-sthetized mice was carried out according to the technique of Hylden and Wilcox (10). IL-la, IL-lra, or one of the antisera was injected into the spinal subarachnoid space in a volume of 5 ~1 (using a Hamilton microsyringe) via a 2X gauge needle inserted between lumbar vertebrae 5 and 6. When required, ILlra was co-injected with IL-la in a total volume of 5 ~1, and SP antiserum or SOM antiserum (each in 5 ~1) was i. t. injected 5 min before the i.t. injection of IL-la. Behavioral observations: For about 1 h before the experiment, and for at least 1 h after the i.t. injections, mice were housed individually in a transparent plastic cage (22.0 x 15.0 x 12.5 cm). The total time spent biting or licking the hindpaws in the 60-min immediately after the i.t. injection of IL-la was taken as the nociceptive response. 1.t. injection of saline provided the control value. Formalin test: An aliquot (20 ~1) of 2 % formalin was injected subcutaneously into the footpad of the right hindpaw, and each mouse was immediately returned to its cage. The total time spent licking and biting the injected hindpaw was measured over a period of 30 min. Data anafy sis : The data are expressed as the mean r S.E.M. of the total time spent biting and licking. Statistical analysis was performed using Dunnett’s multiple comparison test after an analysis of variance (ANOVA). Differences were considered to be significant when P values were less than 0.05. Results of IL-I a: As shown in Fig. 1, Nociceptive responses induced by i. t. injection introduction of IGla into the spinal subarachnoid space of mice by injection between lumbar vertebrae 5 and 6 dose-dependently induced behaviors resembling those occurring in mice injected with formalin (see below). Following such injections, the mice exhibited hindlimb biting and licking of the hindpaws. A significant effect was seen at a dose of as little as 3x 10m4fmol(5.4 fg)/mouse. The time course of these behavioral responses to ILla is shown in Fig.2. The licking and biting response was apparent at 10 min, peaked at 20-40 min, and had disappeared at 60 min.

251

Nwieeptive Responses to Intrathecal IL-1

Vol. 65, No. 3, 1999

The licking and biting response induced by 3x10” fmol/mouse of ILla (total 161 f 15 set) was significantly inhibited by morphine when this was intraperitoneally injected 5 min before the i.t. injection of ILla. In fact, the licking and biting responses in mice given 0.125 or 0.25 mg/kg of morphine were 88 2 22 and 53 + 14 set, respectively (each value being significantly less than the above value for morphine-non-treated mice; P~0.01). Effect of IL-lra on IL-1 a-induced nociceptive responses: IGlra dose-dependently inhibited the behavioral response induced by 3~10.~ fmol(5.4 fg)/mouse of IL-la (Fig.3), with 0.3 fmol(7.2 pg)/mouse of IL-lra almost completely inhibiting the IL-la-induced response. Injection of IL-lra alone at these doses induced neither licking nor biting behavior (data not shown). Effect of antisera against SP and SOM on IL-1 a-induced nociceptive responses: 1.t. injection of SP antiserum significantly suppressed the behavioral responses induced by 3~10~~ fmol/mouse of IL-la (Fig.4). The suppression was about 50 % when 1:32-diluted SP antiserum was injected. However, SOM antiserum produced no significant effect (Fig.4). 300 ** f ; E ‘3 P z z z m

200 -

100 -

P 3 j O_ saline

3x10

a

3x10

4

3

3x10’

IL-1 Q ( fmol )

Fig.1 Dose-dependent induction of nociceptive responses by i.t. injection Values are mean * S.E.M. (n=lO). **P
of IL-la.

Effect of IL-Ira on formalin-induced nociceptive responses: Injection of 2 % formalin into the footpad induced a biphasic nociceptive response consisting of scratching, and licking and biting of the injected hindpaw: a rapid phase at O-5 min and a slower phase at lo-30 min (14,15). Nthough IL-lra did not affect the rapid phase response, it reduced the slow phase response partially, but significantly, at a dose of 30 pmol(720 ng)/mouse or more (Fig.5). Discussion In the present study on mice, we found that i.t. injection (between lumbar vertebrae 5 and 6) of ILla, at as little as 3~10~ fmol (5.4 fg)/mouse, induced licking and biting of the unstimulated hindpaws. Co-injection of IL-la and IL-lra decreased the licking and biting behaviors. In addition, the licking and biting behaviors were significantly suppressed (by 50 % or so) by i.t. injection of an antiserum against SP. These behaviors induced by IL-la were inhibited by morphine, indicating that they are nociceptive responses. Moreover, the nociceptive behaviors induced by formalin injection into the footpad of mice were also shown to be significantly inhibited by 30 pmol (720 ng)/mouse of IL-lra.

258

Nociceptive Responseto Intratheca L-1

Vol. 65, No. 3 1999

125 -

z 0

100’

z ._

75-

i? E .a P 5

50’

T

0

10

20

30

40

Time after injection

50

60

( min )

Fig.2 Time course of nociceptive responses to ILlcx. Effects of various i.t. doses of ILla are shown: 30 fmol(O), 3 fmol(O), 3~10~ fmol (A), 3~10.~ fmol (X), Saline (0). Each value is the mean f S.E.M. (n=lO) of the total time spent on licking and biting behaviors in a given 10 min period (ending at the time shown). *P
150

100

50

0 IL-1 a (fttIOl) IL-1 ra ( fmol )

saline 0

3x104 0

3x104 3X10-2

3x10” 1.5 x10-1

3x104 3x10”

Fig.3 Effect of ILlra on ILl-induced nociceptive responses. Each indicated dose of ILlra was co-injected i.t. with 3~10~ fmol of ILla. Values are mean + S.E.M. (n=lO). **P
Lipopolysaccharide is known to stimulates the production and/or release of various kinds of cytokines (16 . Meller et al. (11) have demonstrated that i .t . injection into rats of either a co-injection of IL1 B (10 ng) and interferon y (1000 U) or a Iipopolysaccharide (150 pg) induces hyperalgesia. However, in the present study we found that a very low dose of ILla itself can induce nociceptive responses.

259

Nockqtive Responses to INmtkcal JL-1

Vol. 65, No. 3, 1999

saline

Saline

1: 512

1: 128

1:32

SP sntlserum

sallne

IL-1 a 3 X104

1: 128

1: 3i

SOM antlserum (hOI)

Fig.4 Effect of antisera against SP and SOM on ILla-induced nociceptive responses. Each antiserum diluted as indicated was i.t. injected 5 min before i.t. injection of 3x10’ fmol of IL-la. Values are mean + S.E.M. (n=lO). *P
“‘1

T

A

Q

150 -

V

“E

‘L

e ._

100’

.Z P II

5

z ._

50’

5 ._ J

0‘

saline

15

7.5

30

60

IL-1 ra ( pm01 )

Fig.5 Effect of IGlra on formalin-induced nociceptive response. Each indicated dose of IL-lra was i.t. injected 5 min before subcutaneous injection of 2 % formalin into the footpad. Values are mean * S.E.M. (n=lO). *P
260

Neeieeptive Response to Intratheeal IL-1

Vol. 65, No. 3 1999

It has been reported that IL-1 is synthesized by astrocytes and microglia in the rat brain (17). It has also been shown that IL1 stimulates the pr~uction of SP in inflamed tissues (18) in chro~~f~ cells (19), and in the myenteric plexus (20). Cultured sympathetic neurons have been shown to produce IL-I, and IL-l accelerates the production of SP from the same neurons (21). It has also been shown that IL1 stimulates the release of SP from the rat isolated spinal cord (22). The role of SP as a neurotransmitter in spinal pain pathways has already been established. Indeed, SP is released from the spinal cord after noxious peripheral stimulation (23,24). Moreover, the mRNA for IL-l receptors is detectable in neurons within the rat spinal cord (25). Although we did not measure SP in the present study, the results mentions above strongly suggest that i. t. injection of IL-1 might lead to a production or release of SP in spinal cord, and that IL1 is involved in the function of spina cord. Interestingly, SP-induced hyperalgesia has been shown to be suppressed by IL-lra (26), suggesting that IL-1 and SP may mutually act to augment their actions. SOM is also an established neurotransmitter in the spinal cord, and IL-1 has been shown to stimulate the synthesis of SOM in rat brain and also its release from the hy~~~~us (27). However, in our present study, we could not detect any incaution by SOM to the indu~on of nociceptive behaviors by IL-la. Since the SOM antiserum used showed a tendency to inhibit the effect of IL-la, we might be able to obtain a positive result by using a more potent antiserum. While our study was in progress, Watkins et al. (12) reported that the i.t. injection of IL-ha (50 or 100 pg) into rats suppressed the hyperalgesia induced by the injection of formalin into the footpad (using the tailflick response to radiant heat as the test response). Although their doses of IL-lra are much higher than those we used, this seems to be due to that they injected hurnurz re~mbin~t ILl-ra into rui?s. It has been shown that SP is released from dorsal horn following the injection of formalin into the hindpaw of mice (14). Although we have no available data indicating the release of IL-1 in spinal cord, our results (an inhibition of formalin-induced nociceptive responses by IL-lra) together with those mentioned above suggest that IL1 and IL-l-induced release of SP may be involved, at least in part, in formalin-induced hyperalgesia. On the basis of the above reports and our present findings, it seems likely that IL-I plays a role in the pain pathways running through the spinal cord by directly or indirectly releasing SP. Our results support the idea put forward by Meller et al. (11) and Watkins et al. (12) that the IL1 produced or released (possibly) by glia cells has an important role in hyperalgesia as an augumentor of the actions of known neurotransmitters in the spinal cord. If true, such a role for IL1 in the spinal cord would seem to be an analogue of the roles played by IL-1 in the self-defence mechanisms already demonstrated in a variety of fields (16). These include immune reactions (16), ideation (16), hematopoi~is (16), gastric acid secretion (28), regulation of blood glucose (29), the microv~cul~ circulation (30) and muscle fatigue (31).

1: 3. 4. 5. 6. 7. 8.

References T. OKA, S. AOU and T. HORI, Brain Res. 624 61-68 (1993). K.YABUUCHI, A. NISHIYORI, M. MASABUMI and M. SATOH, Eur. .I. Pharmacol. 300 59-65 (1996). H. NAKAMURA, K. NAKANISHI, A. KITA and T. KADOKAWA, Eur. J. Pharmacol. 149 49-54 (1988). S.H. FERREIRA, B.B. LQRENZETII, A.F. BRISTOW and S. POOLE, Nature 334 698700 (1988). S.H. FERREIRA, B.B. LORENZEZTI and S. POOLE, Br. J. Pharmacol. 110 1227-1231 (1993). M.N. PERKINS and D. KELLY, Neuroph~~logy 33 6.57-660 (1994). H. FUKUOKA, M. KAWATANI, T. HISAMITSU and C. TAKESHIGE, Brain Res. 657 133-140 (1994). S. SKILLING. D.H. SMULLIN. A.J. BEITZ and AA. LARSON. J. Neurochem. 51 127” 132 (1988). ’

9. D.H. SMULLIN, S.R. SKILLING and A.A. LARSON, Pain 42 93-101 (1990). 10. J.L.K. EfYLDEN and G.L. WILCOX, Brain Res. 217 212-215 (1980). 11. S.T. h&iLLER, C. DYCSTRA, D. GRZYBYKI, S. MURPHY and G.F. GEBHART, Neuroph~acoiogy

33 1471-1478 (1994).

Vol. 65, No. 3, 1999

12. L.R.WATKINS, 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

Nociceptive Responses to Inbathed IL-1

261

D. MARTIN, P. ULRICH, K.J. TRACEY and S.F. MAIER, Pain 71 225235 (1997). N. FUJIOKA, N. MUKAIDA, A. HAEtAD.4, M. AKIYAMA, T. KASAHARA, K. KUNG, A. 001, M. MAI and K MATSUSHIMA, J. Leukocyte Biol. 58 90-98 (1995). R.M. KANTNER, B.D. GOLDSTEIN and M.L. KIRBY, Brain Res 385 282-290 (1986). T. SAKURADA, K. KASTUMATA, H. YOGO, K. TAN-NO, S. SAKURADA, M. OHBA and K. KISARA, Pain 60 175-180 (1995). C.A. DINARELLO, Blood 77 1627-1652 (1991). R.M. LECHAN, R. TONI, B.D. CLARK, J.G. CANNON, AR. SHAW, C.A. DINARELLO and S. REICHLIN, Brain Res. 514 135-140 (1990). M. PERRETTI, A. AHLUWALIA, R.J. FLOWER and S. MANZINI, Immunology 80 73-77 (1993). R.L. ESKAY, L.E. EIDEN and C. HSU, Endocrinology 130 2252-2258 (1992). S.M. HURST, AM. STANISZ, K.A. SHARKEY and S.M. COLLINS, Gastroenterology 105 1754-1760 (1993). M. FREIDIN, M.V.L. BENNETT and J.A. KESSLER, Proc. Natl. Acad. Sci. U.S.A 89 10440-10443 (1992). M. MALCANGIO, N.G. BOWERY, R.J. FLOWER and M. PERRE’ITI, Eur. J. Pharmacol. 299 113-118 (1996). 134-140 (1986). A.W. DUGGAN and I.A. I-IENDRY, Neurosci. Lett. 68 Y. KURAISHI, N. HIROTA, Y. SATO, N. HANASHIMA, H. TAKAGI and M. SATO, Neuroscience 30 241-250 (1989). Brain Res. Bull. 42 463-467 D. GAYLE, S.E. ILYIN and C.R. PLATA-SAL&MAN, (1997). A.J. DAVIS and M.N. PERKINS, Br. J. Pharmacol. 118 2206-2212 (1996). D.E. SCARBOROUGH, S.L. LEE, C.A. DINARELLO and S. REICHLIN, Endocrinology 124 549-551 (1989). Y. TACHE and E. SAPERAS, Ann. New York Acad. Sci. 664 353-368 (1992). Y. END0 and M. NAKAMURA, Gen. Pharmacol. 24 1039-1053 (1993). Y. ENDO, M. NAKAMURA, Y. NITI’A and K. KUMAGAI, Br. J. Pharmacol. 114 187-193 (1995). Y. ENDO, T. TABATA, H. KURODA, T. TADANO, K. MATSUSHIMA and M. WATANABE, J. Physiol. 509 587-598 (1998).