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Hyperalgesia induced by low doses of thymulin injections: possible involvement of prostaglandin E2
Journal of Neuroimmunology 73 Ž1997. 162–168
Hyperalgesia induced by low doses of thymulin injections: possible involvement of prostaglandin E 2 B. Safieh-Garabedian
a,)
, S.A. Kanaan a , R.H. Jalakhian a , S. Poole c , S.J. Jabbur b, N.E. Saade´ b
a
b
Department of Biology, Faculty of Arts and Sciences, American UniÕersity of Beirut, P.O. Box 11-0236, Beirut, Lebanon Departments of Human Morphology and Physiology, Faculty of Medicine, American UniÕersity of Beirut, P.O. Box 11-0236, Beirut, Lebanon c DiÕision of Endocrinology, National Institute for Biological Standards and Control, Blanche Lane, Potters Bar, Herts EN6 3QG, UK Received 4 June 1996; revised 19 September 1996; accepted 23 September 1996
Abstract Thymulin injection into rats Ž20–150 ng. i.p. caused a significant reduction in both mechanical Žpaw pressure test. and thermal Žhot plate and tail flick tests. nociceptive thresholds. Thymulin injection also doubled IL-1 b level in the liver of these animals. Induced hyperalgesia was reversed completely by a-MSH related tripeptide, Lys– D-Pro–Val in low doses, which is known to antagonize IL-1 b and PGE 2 induced hyperalgesia, but was only partly reversed by IL-1 b related tripeptide, Lys– D-Pro–Thr at high doses, which is known to antagonize IL-1 b induced hyperalgesia only. We conclude from these results that thymulin causes hyperalgesia and that this effect is at least in part mediated via PGE 2 and its effectiveness at low concentration implies a physiological role for this thymic hormone. Keywords: Thymulin; Hyperalgesia; Interleukin-1 b ; Prostaglandin; Neuroimmunology
1. Introduction There is considerable evidence for the existence of bi-directional immune–neuro-endocrine interactions, representing an important homeostatic mechanism in the body ŽBesedovsky and Del Rey, 1996.. Cytokines and other products of immunocompetent cells are known to play a crucial role as signaling molecules in these interactions ŽBlalock, 1994; Madden and Felten, 1995. and this has led to the proposal that the immune system is capable of functioning as a diffuse sense organ informing the brain about events occurring at the periphery ŽKent et al., 1992; Maier et al., 1994.. The thymus gland produces several peptide hormones, whose release are under neuro-endocrine control ŽKendall, 1991; Kendall and Stebbings, 1994.. One such hormone, thymulin ŽBach et al., 1976., known to be involved in immunomodulation ŽSafieh-Garabedian et al., 1992., is a highly conserved nonapeptide produced within the thymus gland by two discrete populations of epithelial cells located in the subcapsularrperivascular cortex and medulla, respectively ŽDardenne et al., 1977; ) Corresponding author. Tel.: q961-1-350000 ext. 3913; fax: q961-12124781995; e-mail: [email protected]
Kendall et al., 1991.. Thymulin is readily detectable in the blood of humans, rats and a variety of species ŽDardenne et al., 1977; Safieh et al., 1990. and requires zinc to express its biological activity ŽDardenne et al., 1993.. It has two major actions on T-cells and their immature precursors: that of induction of differentiation markers and enhancement of various T-cell and NK cell activities ŽIncefy et al., 1980; Bordigoni et al., 1984.. The high affinity binding sites for thymulin present on human acute lymphoblastic T-cell lines, that have immature thymocyte phenotype, strongly suggests that this hormone may be important in the early stages of intra-thymic T-cell development ŽPleau ´ et al., 1980.. Furthermore, recent work has indicated that thymulin has a modulatory effect on cytokine release by human peripheral mononuclear cells ŽSafiehGarabedian et al., 1993.. Although little is known about the role of the immune system in mediating events that can also result in hyperalgesia ŽWatkins et al., 1995., it has been recently established that pro-inflammatory cytokines Žproduced by immunocompetent cells. like tumour necrosis factor-a ŽTNFa . and interleukin-1 b ŽIL-1 b ., can be involved ŽFerreira, 1993; Cunha et al., 1992.. Recently, Safieh-Garabedian et al. Ž1995. have demonstrated that IL-1 b receptor antago-
0165-5728r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 6 . 0 0 1 9 5 - 6
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nist significantly reduced hyperalgesia in rats, caused by complete Freund’s adjuvant. More recently, we have developed an animal model for localized inflammatory hyperalgesia following intraplantar endotoxin injections in the hind paw of rats and mice ŽKanaan et al., 1996. and shown, using this model, that thymulin injections Ži.p.. at supraphysiological levels significantly reduced both thermal and mechanical hyperalgesia ŽSafieh-Garabedian et al., 1996.. In this study we have investigated the effect of low level injection of thymulin on the generation of hyperalgesia, by utilizing both mechanical Žpaw pressure latency. and thermal Žhot plate latency and tail flick latency. tests for nociceptive thresholds in rats. We have further investigated the possible reversal of thymulin induced hyperalge-
Fig. 2. The time courses of thermal hyperalgesia as assessed by the HP test, induced by 50 ng thymulin injection Ži.p.. in rats Žtop panel.. Maximum hyperalgesia Žat 6 h. induced by different doses of thymulin injections Žbottom panel..
sia by an a-MSH related tripeptide, Lys– D-Pro–Val, known to antagonize the effects of IL-1 b and prostaglandin E 2 ŽPGE 2 . induced hyperalgesia ŽPoole et al., 1992. and Lys– D-Pro–Thr, a peptide related to IL-1 bŽ193–195., which is known to antagonize IL-1 b evoked hyperalgesia only ŽFerreira et al., 1988..
2. Materials and methods Fig. 1. The time course of mechanical hyperalgesia induced by 50 ng thymulin injection Ži.p.. in rats Žtop panel.. Maximum hyperalgesia Žat 6 h. induced by different doses of thymulin injections Žbottom panel.. The degree of significance of differences is compared to values in saline injected animals. This comparison is also used in Figs. 2 and 3. Data on dose response effects of thymulin are presented in this figure and in Figs. 2 and 3 as % variation from the baseline for easier comparison of the effects on the various pain tests.
2.1. Animals Adult male Sprague-Dawley rats ŽCharles River, Celco, Italy. were used in all the experiments Ž5–6 per group.. The animals Ž150–250 g. were housed under optimum conditions of light and temperature Ž12 h light and 12 h dark cycle; 22 " 38 C., with food and water provided ad
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libitum. All experiments were carried out with strict adherence to ethical guidelines ŽZimmerman, 1983.. 2.2. BehaÕioral measurements Thermal and mechanical pain tests were performed for 3 consecutive days prior to any injections to establish a constant baseline. The paw pressure test ŽPP. was used to assess mechanical hyperalgesia and the hot plate ŽHP. and tail immersion tests ŽTF. were performed for the assessment of thermal hyperalgesia, as described in detail previously ŽKanaan et al., 1996.. Briefly, for the HP test, animals were individually put on a hot surface plate Ž52.8– 53.48C. and the latency of the first sign of paw licking or jumping to avoid the heating pain was taken as an index of the pain threshold. For the TF test, the tails were immersed into a beaker of distilled water ŽT s 50.58C. and the withdrawal latency for tail flicking was recorded. MechanFig. 4. IL-1 b levels in the liver for different groups of rats Ž ns 5 each. at different time intervals, following 50 ng thymulin injections Ži.p.., as compared to naive and saline injected animals Žafter 1 h.. The degree of significance is compared to values in naive animals.
ical hyperalgesia was measured by the paw pressure test ŽPP., by applying a constant pressure of 0.20 kgrcm2 alternately to the left and right hind paws with a 5 min interval between consecutive applications. The pressure was discontinued when the animals displayed a typical pain reaction characterized by a vigorous flexion reflex. 2.3. Experimental protocols
Fig. 3. The time course of thermal hyperalgesia as assessed by the TF test, induced by 50 ng thymulin injection Ži.p.. in rats Žtop panel.. Maximum hyperalgesia Žat 6 h. induced by different doses of thymulin injections Žbottom panel..
Thymulin ŽSigma, St. Louis, MO, USA. was injected intraperitoneally Ži.p.. into various groups of rats and each rat in each group Ž n s 5–6. received the same dose of either 5, 20, 150 or 1000 ng dissolved in 100 m l physiological saline. In a control group Ž n s 5. each rat received 100 m l saline injection only. After establishing the pain test baseline values, as described above, pain tests were carried out at 3, 6, 9 and 24 h after thymulin injection. In another set of experiments, separate rat groups Ž n s 5 in each group. received i.p. injection of one of the two tripeptides of Lys– D-Pro–Val or Lys– D-Pro–Thr, custom synthesized by Cambridge Research Biochemical ŽCambridge, UK.. In one group Ž n s 5., each rat received 5 mgrkg Ži.p.. of Lys– D-Pro–Val 30 min before the injection of thymulin. In other rat groups Ž n s 5 in each group., the response to different doses Ž0.2 or 1 mgrkg. of Lys– D-Pro–Val was tested at 6 h which coincided with the time of peak hyperalgesia from thymulin. The other tripeptide, Lys– D-Pro–Thr, was injected i.p. Ž60 mgrkg. 30 min prior to thymulin injection in a group of rats Ž n s 5. and the time course of the effects was observed at 3, 6, 9 and 24 h post thymulin injection. In two other groups Ž n s 5, each. the response to different doses Ž10 or 30 mgrkg. of
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Lys– D-Pro–Thr was tested at 6 h. In control groups Ž n s 5 each., rats received one of the tripeptides alone Žin 100 m l saline. and the various pain tests were carried out at 3, 6, 9 and 24 h after the injection. The doses used for these two tripeptides were based on previous data established by Ferreira et al. Ž1988. and Poole et al. Ž1992.. 2.4. IL-1b assay The level of IL-1 b was measured in the liver since this organ, is known to contribute significantly, together with blood mononuclear cells, to cytokine production in response to inflammation and has been shown to be involved in the signaling pathway to the brain in sickness induced behavior Že.g. fever, hyperalgesia and anorexia. by Watkins et al. Ž1994.. The liver was removed from a group Ž n s 5. of rats that received no treatment and from groups of rats Ž n s 5–6 in each group. at 1, 2 and 4 h after 50 ng thymulin injection. Liver was also removed at 1 h from a group of rats Ž n s 5. that received 100 m l saline injection only. Liver tissue was homogenized in phosphate buffered saline ŽPBS. containing 0.4 M NaCl, 0.05% Tween-20, 0.5% bovine serum albumin, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM
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benzethonium chloride, 10 mM EDTA and 20 KIrml aprotinin. The homogenates were centrifuged at 12 000 = g for 60 min at 48C. IL-1 b content in the supernatant was measured by two-site enzyme-linked immunoassay ŽELISA. as detailed previously ŽSafieh-Garabedian et al., 1995., using immunoaffinity purified polyclonal sheep anti-rat IL-1 b , ŽTaktak et al., 1991. to coat high binding microtiter plates. Recombinant rat IL-1 b Ža generous gift from Dr. Robert Newton, DuPont-Merck, Wilmington, DE, USA. was used as the standard and a biotinylated, immunoaffinity purified polyclonal sheep anti-rat IL-1 b as a recognition antibody. The color was developed by using streptavidin horseradish peroxidase ŽDako. and the chromagen, 3,3X ,5,5-tetramethyl-benzidine ŽSigma. and the optical density ŽOD. measured at 450 nm.
2.5. Data analysis All results are given as mean " standard error of the mean. The degree of significance of differences between experimental groups was performed by the ANOVA test, using the Graph Pad Software version 1.13 and Prism version 1.
Fig. 5. Time course of hyperalgesia induced by 50 ng thymulin injections Ži.p.. and its reversal by i.p. injections of Lys– D-Pro–Val in different pain tests ŽŽA., ŽB. and ŽC... The histograms ŽŽD., ŽE. and ŽF.. illustrate the effects of different doses of Lys– D-Pro–Val measured at 6 h after thymulin injections.
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3. Results Both thymulin and saline injection had no apparent effect on body temperature, eating habit and walking pattern of the animals. Furthermore, rats in the control groups showed stable thresholds for all three pain tests before and after receiving saline injections. 3.1. Effect of thymulin on the pain tests 3.1.1. PP test Thymulin Ž50 ng. significantly Ž P - 0.01. reduced PP latency in the hind paw 3 h after its injection Ž1.61 " 0.03 s; n s 5. when compared with saline controls Ž1.98 " 0.06 s; n s 5. ŽFig. 1A.. Maximum decrease in latency was obtained at 6 h Ž1.15 " 0.05 s; n s 5., which was highly significant Ž P - 0.001. when compared with saline values Ž1.86 " 0.07 s; n s 5.. Mechanical hyperalgesia was still apparent at 9 h Ž P - 0.001. but at 24 h there was no difference between thymulin injected animals and saline controls ŽFig. 1A.. Although the mechanical hyperalgesia was maximal at 6 h following thymulin injection at a dose of 50 ng, significant decrease in PP latencies Ž P - 0.01.
were also evident with lower Ž20 ng. and higher doses Ž150 ng. when tested at 6 h ŽFig. 1B.. At concentrations higher than 150 ng thymulin had no effect on mechanical hyperalgesia ŽFig. 1B..
3.1.2. HP test Thymulin Ž50 ngr100 m l. significantly Ž P - 0.001. reduced HP latency at 3 h after its injection Ž6.67 " 0.54 s; n s 5., when latency values were compared with the saline injected animals Ž10.52 " 0.42 s; n s 5.. Maximum decrease in latency was obtained at 6 h Ž5.42 " 0.44 s; n s 5. which was highly significant Ž P - 0.001. when values were compared with saline controls Ž9.85 " 0.17 s; n s 5.. Thermal hyperalgesia was still apparent at 9 h Ž P - 0.001. but at 24 h there was no difference between thymulin injected animals and saline controls ŽFig. 2A.. Thymulin at 20 ng produced maximal effect Ž P - 0.001.; significant hyperalgesia was also obtained with thymulin at the 50 ng Ž P - 0.01. and at the 150 ngr100 m l ŽP - 0.05. injection doses. At concentrations higher than 150 ngr100 m l thymulin had no effect on thermal hyperalgesia ŽFig. 2B..
Fig. 6. Time course of the effect of Lys– D-Pro–Thr on thymulin induced hyperalgesia Ž50 ng; i.p.. as assessed by different pain tests ŽA–C.. The significance of differences is compared to values of thymulin injections. The histograms ŽD–F. illustrate the effects of different doses of Lys– D-Pro–Thr measured at 6 h after thymulin injections.
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3.1.3. TF test Thymulin Ž50 ngr100 m l. significantly Ž P - 0.001. reduced TF latency at 3 h after its injection Ž2.87 " 0.05 s; n s 5., when latency values were compared with the saline injected animals Ž3.48 " 0.08 s; n s 5.. Maximum decrease in latency was obtained at 6 h Ž2.58 " 0.06 s; n s 5. which was highly significant Ž P - 0.001. when values were compared with saline controls Ž3.46 " 0.10 s; n s 5.. Thermal hyperalgesia was still apparent at 9 h Ž P - 0.01. but at 24 h there was no significant difference between thymulin injected animals and saline controls ŽFig. 3A.. Thymulin at 20 ng and 50 ng was equally effective Ž P - 0.001. at inducing the hyperalgesia, whereas higher doses tested had no significant effect, when values were compared with saline injections ŽFig. 3B.. 3.2. Effect of thymulin on IL-1b leÕel in the liÕer After 1 h of thymulin Ž50 ng. injection there was a significant Ž P - 0.001. increase in the level of IL-1 b in the liver Ž10.09 " 0.84 pgrmg tissue; n s 6., when compared with values obtained from naıve ¨ animals Ž5.04 " 0.54 pgrmg tissue; n s 5. or from rats after 1 h saline injection only Ž4.01 " 0.74 pgrmg tissue; n s 5.. The level of this cytokine remained significantly Ž P - 0.05. elevated at 2 h Ž8.59 " 0.96; n s 5. and returned to a baseline value at 4 h ŽFig. 4.. 3.3. Effect of injection of the tripeptides Lys– D-Pro–Val and Lys– D-Pro–Thr Intraperitoneal injection of Lys– D-Pro–Val at 5 mgrkg completely reversed thymulin induced hyperalgesia ŽFig. 5.. The time course of this effect is given for PP, HP and TF tests in Fig. 5A, B and C, respectively. The dose response results of the tripeptide at 0.2, 1 and 5 mgrkg are shown in Fig. 5D, which also shows that PP test was also reversed completely at the 1 mgrkg dose. Using thymulin induced hyperalgesia as assessed by the various pain tests, Lys– D-Pro–Thr Žthe IL-1 b antagonist. injection showed no effect at low doses Ž1 and 10 mgrkg., but resulted in partial reduction of hyperalgesia at higher doses Ž30 and 60 mgrkg. as illustrated in Fig. 6.
4. Discussion Our results clearly demonstrate that i.p. injection of thymulin in low doses in rats causes hyperalgesia, with significant reduction in both thermal and mechanical nociceptive thresholds. The effect of thymulin was apparent at doses ranging between 20 and 150 ng and was maximal at 50 ng. Thymulin above or below these values had no effect on any of the pain tests performed ŽFig. 1B, Fig. 2B and Fig. 3B.. In a previous work however, we have demonstrated that thymulin at concentrations above 150 ng re-
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duces endotoxin induced hyperalgesia in rats and mice ŽSafieh-Garabedian et al., 1996.. These findings correlate well with reports showing that thymulin in low concentrations enhances the immune responses whereas opposite effects were produced with higher supraphysiological doses ŽRitter and Crispe, 1992.. In line with these observations, Safieh-Garabedian et al. Ž1993. have shown that thymulin at low concentrations stimulates Il-1 b release from human peripheral blood mononuclear cells, whereas at high concentrations it suppresses the release of IL-1 b as well as IL-2, IL-6 and TNF-a . Although thymulin doubled IL-1 b level in the liver 1 h after its injection ŽFig. 4., this effect is milder and less sustained than the changes in IL-1 b level in the skin following intraplantar endotoxin injection Ž10-fold increase in IL-1 b level; unpublished data.. However, the endotoxin induced hyperalgesia was comparable in intensity and duration ŽKanaan et al., 1996. to that produced by thymulin. Furthermore, high levels of IL-1 b Ž15-fold. in the skin were also reported following complete Freund’s adjuvant hyperalgesia ŽSafieh-Garabedian et al., 1995.. The comparisons just cited about the transient and relatively mild increases in IL-1 b level, correlate with our finding that Lys– D-Pro–Thr, at high doses only, partially reversed thymulin induced hyperalgesia. This tripeptide has been shown to antagonize IL-1 b , but not PGE 2 induced hyperalgesia ŽFerreira et al., 1988.. On the other hand, Lys– DPro–Val in doses as low as 1–5 mgrkg reversed almost completely thymulin induced hyperalgesia. This a-MSH related tripeptide, has been recently shown to antagonize both IL-1 b and PGE 2 induced hyperalgesia in rats ŽPoole et al., 1992.. This suggests that PGE 2 plays a much more significant role than IL-1 b in thymulin induced hyperalgesia. This is further suggested by the studies showing that thymulin in low concentrations induced PGE 2 synthesis in cultured mononuclear cells ŽGualde et al., 1982. and thymocytes ŽRinaldi-Garaci et al., 1985.. The role of prostaglandins in pain has been studied extensively since the demonstration of its hyperalgesic action in humans ŽFerreira, 1972.. Recently it has been shown that PGE 2 sensitizes rat sensory neurons to mechanical and thermal stimuli by facilitating neuropeptide release from these neurons ŽHingtgen et al., 1995.. In conclusion, thymulin causes hyperalgesia in rats which appears to be mediated in part via PGE 2 and its effectiveness at low concentration implies a physiological role. Thymulin may well form part of the immune activation system, resulting from illness responses and pathological pain states, which also activate the hypothalamuspituitary axis. Stress hormones ŽBuckingham et al., 1992. as well as other hormones of the neuroendocrine system ŽKendall and Stebbings, 1994. can stimulate thymulin release. Our research continues towards determining whether the hyperalgesic action of thymulin constitutes part of the physiological responses in the overall communication between the immune and the nervous systems.
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Acknowledgements The authors thank John Haddad, Pamela Abou Jaoude and Riad Maalouf for their technical assistance in this study. This research was funded in part by the University Research Board, Diana Tamari Sabbagh Fund and Lebanese National Council for Scientific Research.
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Hyperalgesia induced by low doses of thymulin injections: possible involvement of prostaglandin E 2 B. Safieh-Garabedian
a,)
, S.A. Kanaan a , R.H. Jalakhian a , S. Poole c , S.J. Jabbur b, N.E. Saade´ b
a
b
Department of Biology, Faculty of Arts and Sciences, American UniÕersity of Beirut, P.O. Box 11-0236, Beirut, Lebanon Departments of Human Morphology and Physiology, Faculty of Medicine, American UniÕersity of Beirut, P.O. Box 11-0236, Beirut, Lebanon c DiÕision of Endocrinology, National Institute for Biological Standards and Control, Blanche Lane, Potters Bar, Herts EN6 3QG, UK Received 4 June 1996; revised 19 September 1996; accepted 23 September 1996
Abstract Thymulin injection into rats Ž20–150 ng. i.p. caused a significant reduction in both mechanical Žpaw pressure test. and thermal Žhot plate and tail flick tests. nociceptive thresholds. Thymulin injection also doubled IL-1 b level in the liver of these animals. Induced hyperalgesia was reversed completely by a-MSH related tripeptide, Lys– D-Pro–Val in low doses, which is known to antagonize IL-1 b and PGE 2 induced hyperalgesia, but was only partly reversed by IL-1 b related tripeptide, Lys– D-Pro–Thr at high doses, which is known to antagonize IL-1 b induced hyperalgesia only. We conclude from these results that thymulin causes hyperalgesia and that this effect is at least in part mediated via PGE 2 and its effectiveness at low concentration implies a physiological role for this thymic hormone. Keywords: Thymulin; Hyperalgesia; Interleukin-1 b ; Prostaglandin; Neuroimmunology
1. Introduction There is considerable evidence for the existence of bi-directional immune–neuro-endocrine interactions, representing an important homeostatic mechanism in the body ŽBesedovsky and Del Rey, 1996.. Cytokines and other products of immunocompetent cells are known to play a crucial role as signaling molecules in these interactions ŽBlalock, 1994; Madden and Felten, 1995. and this has led to the proposal that the immune system is capable of functioning as a diffuse sense organ informing the brain about events occurring at the periphery ŽKent et al., 1992; Maier et al., 1994.. The thymus gland produces several peptide hormones, whose release are under neuro-endocrine control ŽKendall, 1991; Kendall and Stebbings, 1994.. One such hormone, thymulin ŽBach et al., 1976., known to be involved in immunomodulation ŽSafieh-Garabedian et al., 1992., is a highly conserved nonapeptide produced within the thymus gland by two discrete populations of epithelial cells located in the subcapsularrperivascular cortex and medulla, respectively ŽDardenne et al., 1977; ) Corresponding author. Tel.: q961-1-350000 ext. 3913; fax: q961-12124781995; e-mail: [email protected]
Kendall et al., 1991.. Thymulin is readily detectable in the blood of humans, rats and a variety of species ŽDardenne et al., 1977; Safieh et al., 1990. and requires zinc to express its biological activity ŽDardenne et al., 1993.. It has two major actions on T-cells and their immature precursors: that of induction of differentiation markers and enhancement of various T-cell and NK cell activities ŽIncefy et al., 1980; Bordigoni et al., 1984.. The high affinity binding sites for thymulin present on human acute lymphoblastic T-cell lines, that have immature thymocyte phenotype, strongly suggests that this hormone may be important in the early stages of intra-thymic T-cell development ŽPleau ´ et al., 1980.. Furthermore, recent work has indicated that thymulin has a modulatory effect on cytokine release by human peripheral mononuclear cells ŽSafiehGarabedian et al., 1993.. Although little is known about the role of the immune system in mediating events that can also result in hyperalgesia ŽWatkins et al., 1995., it has been recently established that pro-inflammatory cytokines Žproduced by immunocompetent cells. like tumour necrosis factor-a ŽTNFa . and interleukin-1 b ŽIL-1 b ., can be involved ŽFerreira, 1993; Cunha et al., 1992.. Recently, Safieh-Garabedian et al. Ž1995. have demonstrated that IL-1 b receptor antago-
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nist significantly reduced hyperalgesia in rats, caused by complete Freund’s adjuvant. More recently, we have developed an animal model for localized inflammatory hyperalgesia following intraplantar endotoxin injections in the hind paw of rats and mice ŽKanaan et al., 1996. and shown, using this model, that thymulin injections Ži.p.. at supraphysiological levels significantly reduced both thermal and mechanical hyperalgesia ŽSafieh-Garabedian et al., 1996.. In this study we have investigated the effect of low level injection of thymulin on the generation of hyperalgesia, by utilizing both mechanical Žpaw pressure latency. and thermal Žhot plate latency and tail flick latency. tests for nociceptive thresholds in rats. We have further investigated the possible reversal of thymulin induced hyperalge-
Fig. 2. The time courses of thermal hyperalgesia as assessed by the HP test, induced by 50 ng thymulin injection Ži.p.. in rats Žtop panel.. Maximum hyperalgesia Žat 6 h. induced by different doses of thymulin injections Žbottom panel..
sia by an a-MSH related tripeptide, Lys– D-Pro–Val, known to antagonize the effects of IL-1 b and prostaglandin E 2 ŽPGE 2 . induced hyperalgesia ŽPoole et al., 1992. and Lys– D-Pro–Thr, a peptide related to IL-1 bŽ193–195., which is known to antagonize IL-1 b evoked hyperalgesia only ŽFerreira et al., 1988..
2. Materials and methods Fig. 1. The time course of mechanical hyperalgesia induced by 50 ng thymulin injection Ži.p.. in rats Žtop panel.. Maximum hyperalgesia Žat 6 h. induced by different doses of thymulin injections Žbottom panel.. The degree of significance of differences is compared to values in saline injected animals. This comparison is also used in Figs. 2 and 3. Data on dose response effects of thymulin are presented in this figure and in Figs. 2 and 3 as % variation from the baseline for easier comparison of the effects on the various pain tests.
2.1. Animals Adult male Sprague-Dawley rats ŽCharles River, Celco, Italy. were used in all the experiments Ž5–6 per group.. The animals Ž150–250 g. were housed under optimum conditions of light and temperature Ž12 h light and 12 h dark cycle; 22 " 38 C., with food and water provided ad
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libitum. All experiments were carried out with strict adherence to ethical guidelines ŽZimmerman, 1983.. 2.2. BehaÕioral measurements Thermal and mechanical pain tests were performed for 3 consecutive days prior to any injections to establish a constant baseline. The paw pressure test ŽPP. was used to assess mechanical hyperalgesia and the hot plate ŽHP. and tail immersion tests ŽTF. were performed for the assessment of thermal hyperalgesia, as described in detail previously ŽKanaan et al., 1996.. Briefly, for the HP test, animals were individually put on a hot surface plate Ž52.8– 53.48C. and the latency of the first sign of paw licking or jumping to avoid the heating pain was taken as an index of the pain threshold. For the TF test, the tails were immersed into a beaker of distilled water ŽT s 50.58C. and the withdrawal latency for tail flicking was recorded. MechanFig. 4. IL-1 b levels in the liver for different groups of rats Ž ns 5 each. at different time intervals, following 50 ng thymulin injections Ži.p.., as compared to naive and saline injected animals Žafter 1 h.. The degree of significance is compared to values in naive animals.
ical hyperalgesia was measured by the paw pressure test ŽPP., by applying a constant pressure of 0.20 kgrcm2 alternately to the left and right hind paws with a 5 min interval between consecutive applications. The pressure was discontinued when the animals displayed a typical pain reaction characterized by a vigorous flexion reflex. 2.3. Experimental protocols
Fig. 3. The time course of thermal hyperalgesia as assessed by the TF test, induced by 50 ng thymulin injection Ži.p.. in rats Žtop panel.. Maximum hyperalgesia Žat 6 h. induced by different doses of thymulin injections Žbottom panel..
Thymulin ŽSigma, St. Louis, MO, USA. was injected intraperitoneally Ži.p.. into various groups of rats and each rat in each group Ž n s 5–6. received the same dose of either 5, 20, 150 or 1000 ng dissolved in 100 m l physiological saline. In a control group Ž n s 5. each rat received 100 m l saline injection only. After establishing the pain test baseline values, as described above, pain tests were carried out at 3, 6, 9 and 24 h after thymulin injection. In another set of experiments, separate rat groups Ž n s 5 in each group. received i.p. injection of one of the two tripeptides of Lys– D-Pro–Val or Lys– D-Pro–Thr, custom synthesized by Cambridge Research Biochemical ŽCambridge, UK.. In one group Ž n s 5., each rat received 5 mgrkg Ži.p.. of Lys– D-Pro–Val 30 min before the injection of thymulin. In other rat groups Ž n s 5 in each group., the response to different doses Ž0.2 or 1 mgrkg. of Lys– D-Pro–Val was tested at 6 h which coincided with the time of peak hyperalgesia from thymulin. The other tripeptide, Lys– D-Pro–Thr, was injected i.p. Ž60 mgrkg. 30 min prior to thymulin injection in a group of rats Ž n s 5. and the time course of the effects was observed at 3, 6, 9 and 24 h post thymulin injection. In two other groups Ž n s 5, each. the response to different doses Ž10 or 30 mgrkg. of
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Lys– D-Pro–Thr was tested at 6 h. In control groups Ž n s 5 each., rats received one of the tripeptides alone Žin 100 m l saline. and the various pain tests were carried out at 3, 6, 9 and 24 h after the injection. The doses used for these two tripeptides were based on previous data established by Ferreira et al. Ž1988. and Poole et al. Ž1992.. 2.4. IL-1b assay The level of IL-1 b was measured in the liver since this organ, is known to contribute significantly, together with blood mononuclear cells, to cytokine production in response to inflammation and has been shown to be involved in the signaling pathway to the brain in sickness induced behavior Že.g. fever, hyperalgesia and anorexia. by Watkins et al. Ž1994.. The liver was removed from a group Ž n s 5. of rats that received no treatment and from groups of rats Ž n s 5–6 in each group. at 1, 2 and 4 h after 50 ng thymulin injection. Liver was also removed at 1 h from a group of rats Ž n s 5. that received 100 m l saline injection only. Liver tissue was homogenized in phosphate buffered saline ŽPBS. containing 0.4 M NaCl, 0.05% Tween-20, 0.5% bovine serum albumin, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM
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benzethonium chloride, 10 mM EDTA and 20 KIrml aprotinin. The homogenates were centrifuged at 12 000 = g for 60 min at 48C. IL-1 b content in the supernatant was measured by two-site enzyme-linked immunoassay ŽELISA. as detailed previously ŽSafieh-Garabedian et al., 1995., using immunoaffinity purified polyclonal sheep anti-rat IL-1 b , ŽTaktak et al., 1991. to coat high binding microtiter plates. Recombinant rat IL-1 b Ža generous gift from Dr. Robert Newton, DuPont-Merck, Wilmington, DE, USA. was used as the standard and a biotinylated, immunoaffinity purified polyclonal sheep anti-rat IL-1 b as a recognition antibody. The color was developed by using streptavidin horseradish peroxidase ŽDako. and the chromagen, 3,3X ,5,5-tetramethyl-benzidine ŽSigma. and the optical density ŽOD. measured at 450 nm.
2.5. Data analysis All results are given as mean " standard error of the mean. The degree of significance of differences between experimental groups was performed by the ANOVA test, using the Graph Pad Software version 1.13 and Prism version 1.
Fig. 5. Time course of hyperalgesia induced by 50 ng thymulin injections Ži.p.. and its reversal by i.p. injections of Lys– D-Pro–Val in different pain tests ŽŽA., ŽB. and ŽC... The histograms ŽŽD., ŽE. and ŽF.. illustrate the effects of different doses of Lys– D-Pro–Val measured at 6 h after thymulin injections.
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3. Results Both thymulin and saline injection had no apparent effect on body temperature, eating habit and walking pattern of the animals. Furthermore, rats in the control groups showed stable thresholds for all three pain tests before and after receiving saline injections. 3.1. Effect of thymulin on the pain tests 3.1.1. PP test Thymulin Ž50 ng. significantly Ž P - 0.01. reduced PP latency in the hind paw 3 h after its injection Ž1.61 " 0.03 s; n s 5. when compared with saline controls Ž1.98 " 0.06 s; n s 5. ŽFig. 1A.. Maximum decrease in latency was obtained at 6 h Ž1.15 " 0.05 s; n s 5., which was highly significant Ž P - 0.001. when compared with saline values Ž1.86 " 0.07 s; n s 5.. Mechanical hyperalgesia was still apparent at 9 h Ž P - 0.001. but at 24 h there was no difference between thymulin injected animals and saline controls ŽFig. 1A.. Although the mechanical hyperalgesia was maximal at 6 h following thymulin injection at a dose of 50 ng, significant decrease in PP latencies Ž P - 0.01.
were also evident with lower Ž20 ng. and higher doses Ž150 ng. when tested at 6 h ŽFig. 1B.. At concentrations higher than 150 ng thymulin had no effect on mechanical hyperalgesia ŽFig. 1B..
3.1.2. HP test Thymulin Ž50 ngr100 m l. significantly Ž P - 0.001. reduced HP latency at 3 h after its injection Ž6.67 " 0.54 s; n s 5., when latency values were compared with the saline injected animals Ž10.52 " 0.42 s; n s 5.. Maximum decrease in latency was obtained at 6 h Ž5.42 " 0.44 s; n s 5. which was highly significant Ž P - 0.001. when values were compared with saline controls Ž9.85 " 0.17 s; n s 5.. Thermal hyperalgesia was still apparent at 9 h Ž P - 0.001. but at 24 h there was no difference between thymulin injected animals and saline controls ŽFig. 2A.. Thymulin at 20 ng produced maximal effect Ž P - 0.001.; significant hyperalgesia was also obtained with thymulin at the 50 ng Ž P - 0.01. and at the 150 ngr100 m l ŽP - 0.05. injection doses. At concentrations higher than 150 ngr100 m l thymulin had no effect on thermal hyperalgesia ŽFig. 2B..
Fig. 6. Time course of the effect of Lys– D-Pro–Thr on thymulin induced hyperalgesia Ž50 ng; i.p.. as assessed by different pain tests ŽA–C.. The significance of differences is compared to values of thymulin injections. The histograms ŽD–F. illustrate the effects of different doses of Lys– D-Pro–Thr measured at 6 h after thymulin injections.
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3.1.3. TF test Thymulin Ž50 ngr100 m l. significantly Ž P - 0.001. reduced TF latency at 3 h after its injection Ž2.87 " 0.05 s; n s 5., when latency values were compared with the saline injected animals Ž3.48 " 0.08 s; n s 5.. Maximum decrease in latency was obtained at 6 h Ž2.58 " 0.06 s; n s 5. which was highly significant Ž P - 0.001. when values were compared with saline controls Ž3.46 " 0.10 s; n s 5.. Thermal hyperalgesia was still apparent at 9 h Ž P - 0.01. but at 24 h there was no significant difference between thymulin injected animals and saline controls ŽFig. 3A.. Thymulin at 20 ng and 50 ng was equally effective Ž P - 0.001. at inducing the hyperalgesia, whereas higher doses tested had no significant effect, when values were compared with saline injections ŽFig. 3B.. 3.2. Effect of thymulin on IL-1b leÕel in the liÕer After 1 h of thymulin Ž50 ng. injection there was a significant Ž P - 0.001. increase in the level of IL-1 b in the liver Ž10.09 " 0.84 pgrmg tissue; n s 6., when compared with values obtained from naıve ¨ animals Ž5.04 " 0.54 pgrmg tissue; n s 5. or from rats after 1 h saline injection only Ž4.01 " 0.74 pgrmg tissue; n s 5.. The level of this cytokine remained significantly Ž P - 0.05. elevated at 2 h Ž8.59 " 0.96; n s 5. and returned to a baseline value at 4 h ŽFig. 4.. 3.3. Effect of injection of the tripeptides Lys– D-Pro–Val and Lys– D-Pro–Thr Intraperitoneal injection of Lys– D-Pro–Val at 5 mgrkg completely reversed thymulin induced hyperalgesia ŽFig. 5.. The time course of this effect is given for PP, HP and TF tests in Fig. 5A, B and C, respectively. The dose response results of the tripeptide at 0.2, 1 and 5 mgrkg are shown in Fig. 5D, which also shows that PP test was also reversed completely at the 1 mgrkg dose. Using thymulin induced hyperalgesia as assessed by the various pain tests, Lys– D-Pro–Thr Žthe IL-1 b antagonist. injection showed no effect at low doses Ž1 and 10 mgrkg., but resulted in partial reduction of hyperalgesia at higher doses Ž30 and 60 mgrkg. as illustrated in Fig. 6.
4. Discussion Our results clearly demonstrate that i.p. injection of thymulin in low doses in rats causes hyperalgesia, with significant reduction in both thermal and mechanical nociceptive thresholds. The effect of thymulin was apparent at doses ranging between 20 and 150 ng and was maximal at 50 ng. Thymulin above or below these values had no effect on any of the pain tests performed ŽFig. 1B, Fig. 2B and Fig. 3B.. In a previous work however, we have demonstrated that thymulin at concentrations above 150 ng re-
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duces endotoxin induced hyperalgesia in rats and mice ŽSafieh-Garabedian et al., 1996.. These findings correlate well with reports showing that thymulin in low concentrations enhances the immune responses whereas opposite effects were produced with higher supraphysiological doses ŽRitter and Crispe, 1992.. In line with these observations, Safieh-Garabedian et al. Ž1993. have shown that thymulin at low concentrations stimulates Il-1 b release from human peripheral blood mononuclear cells, whereas at high concentrations it suppresses the release of IL-1 b as well as IL-2, IL-6 and TNF-a . Although thymulin doubled IL-1 b level in the liver 1 h after its injection ŽFig. 4., this effect is milder and less sustained than the changes in IL-1 b level in the skin following intraplantar endotoxin injection Ž10-fold increase in IL-1 b level; unpublished data.. However, the endotoxin induced hyperalgesia was comparable in intensity and duration ŽKanaan et al., 1996. to that produced by thymulin. Furthermore, high levels of IL-1 b Ž15-fold. in the skin were also reported following complete Freund’s adjuvant hyperalgesia ŽSafieh-Garabedian et al., 1995.. The comparisons just cited about the transient and relatively mild increases in IL-1 b level, correlate with our finding that Lys– D-Pro–Thr, at high doses only, partially reversed thymulin induced hyperalgesia. This tripeptide has been shown to antagonize IL-1 b , but not PGE 2 induced hyperalgesia ŽFerreira et al., 1988.. On the other hand, Lys– DPro–Val in doses as low as 1–5 mgrkg reversed almost completely thymulin induced hyperalgesia. This a-MSH related tripeptide, has been recently shown to antagonize both IL-1 b and PGE 2 induced hyperalgesia in rats ŽPoole et al., 1992.. This suggests that PGE 2 plays a much more significant role than IL-1 b in thymulin induced hyperalgesia. This is further suggested by the studies showing that thymulin in low concentrations induced PGE 2 synthesis in cultured mononuclear cells ŽGualde et al., 1982. and thymocytes ŽRinaldi-Garaci et al., 1985.. The role of prostaglandins in pain has been studied extensively since the demonstration of its hyperalgesic action in humans ŽFerreira, 1972.. Recently it has been shown that PGE 2 sensitizes rat sensory neurons to mechanical and thermal stimuli by facilitating neuropeptide release from these neurons ŽHingtgen et al., 1995.. In conclusion, thymulin causes hyperalgesia in rats which appears to be mediated in part via PGE 2 and its effectiveness at low concentration implies a physiological role. Thymulin may well form part of the immune activation system, resulting from illness responses and pathological pain states, which also activate the hypothalamuspituitary axis. Stress hormones ŽBuckingham et al., 1992. as well as other hormones of the neuroendocrine system ŽKendall and Stebbings, 1994. can stimulate thymulin release. Our research continues towards determining whether the hyperalgesic action of thymulin constitutes part of the physiological responses in the overall communication between the immune and the nervous systems.
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Acknowledgements The authors thank John Haddad, Pamela Abou Jaoude and Riad Maalouf for their technical assistance in this study. This research was funded in part by the University Research Board, Diana Tamari Sabbagh Fund and Lebanese National Council for Scientific Research.
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