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Failure of intravenous immunoglobulin (IVIg) therapy in experimental autoimmune neuritis (EAN) of the Lewis rat
Journal of Neuroimmunology 76 Ž1997. 112–116
Failure of intravenous immunoglobulin ž IVIg/ therapy in experimental autoimmune neuritis ž EAN/ of the Lewis rat U. Enders, K.V. Toyka, H.-P. Hartung, R. Gold
)
Department of Neurology, Neuroimmunology Branch and Clinical Research Group for Multiple Sclerosis, Julius-Maximilians UniÕersitat, ¨ Josef-Schneider-Straße 11, D-97080 Wurzburg, Germany ¨ Received 2 December 1996; revised 16 January 1997; accepted 17 January 1997
Abstract Experimental autoimmune neuritis ŽEAN. is an animal model for Guillain-Barre´ syndrome ŽGBS.. Intravenous immunoglobulins ŽIVIg. are an effective treatment for GBS, but their mechanism of action is not well understood. Here we tested whether IVIg treatment, a potent modulator of proinflammatory assaults, reduces inflammation in EAN. The evaluation of IVIg treatment failed to demonstrate a salutary effect in different models of EAN. IVIg appears not to suppress the acute inflammatory insult on the peripheral nerve, but may have beneficial long-term effects not looked for in the present investigation. Keywords: Immune globulin; Neuritis; Guillain-Barre´ syndrome
1. Introduction Intravenous administration of 7S-immunoglobulins ŽIVIg. obtained from pools of 10,000 or more donors is now an established therapy in immunopathologic disorders Žreviewed in Kaveri et al., 1991.. In the past decade the use of IVIg has been extended into the therapy of diverse neurological diseases thought to be autoimmune in nature. Recent reports Žvan der Meche et al., 1992. claimed a similar efficacy of high-dose IVIg and plasmapheresis in GBS, the most frequent cause of acute ascending paralysis. The results of the largest ever conducted trial in this disorder, comparing plasmapheresis ŽPE., IVIg and a combination of PE and IVIg, demonstrated equipotency of IVIg ŽPSGBS Group, 1997.. A multitude of potential mechanisms may underlie the beneficial actions of IVIg. Amongst these, inhibition of proinflammatory effects plays a central role. IVIg can act by anti-idiotypic effects on T-cell receptors, inhibition of complement-mediated cytotoxicity, modulation of cytokine production and direct binding of cytokines and blockade of
) Corresponding author. Tel.: q49-931-2012605; fax: q49-9312012697.
Fc receptors Žreviewed in Stangel et al., 1997.. Since systematic studies are not possible in patients we decided to investigate these actions in experimental autoimmune neuritis of Lewis rats, an animal model of GBS ŽHartung et al., 1993; Hartung et al., 1995.. We wished to address the effects of IgG on the different models of EAN and therefore used all three variants in the Lewis rat: EAN induced by active immunization with whole peripheral myelin ŽSmith et al., 1979., the neuritogenic peptide of the P2 protein ŽBrostoff et al., 1977., or by adoptive transfer of activated P2-specific neuritogenic T-lymphocytes ŽATEAN. ŽLinington et al., 1984.. We failed to demonstrate an immunomodulatory effect in either model.
2. Materials and methods 2.1. Animals Female Lewis rats were obtained from Charles River Laboratories ŽSulzfeld, Germany. and were 7–8 weeks old. Animals were housed in plastic cages without grid floors and given commercial food pellets and water ad libidum. All experiments were performed according to the Bavarian state regulations for animal experimentation and approved by the responsible authorities.
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 7 . 0 0 0 3 9 - 8
U. Enders et al.r Journal of Neuroimmunology 76 (1997) 112–116
113
Table 1 Human IgG in rat sera. Results are given as mgrdl Ž n s 6 rats per group. Day 10 Žmean " SD.
Day 15 Žmean " SD.
Controls Standard dose treatment Ž400 mgrkg body weight. High dose treatment Ž2000 mgrkg body weight.
73 " 15 2087 " 179 4736 " 562
145 " 41 1428 " 190 2532 " 161
2.2. Disease induction
paraparesis, 6 s moderate paraparesis, 7 s severe paraparesis, 8 s tetraparesis, 9 s moribund and 10 s death.
For active EAN, Lewis rats were inoculated into the footpad with 50 m l of an emulsion of an equal volume of saline and complete Freunds adjuvant containing 2 mg peripheral nerve myelin prepared from bovine spinal roots ŽNorton and Poduslo, 1973; Uyemura et al., 1972. or 100 m g P2 peptide spanning aa 53–78 of bovine P2 ŽMedprobe, Oslo. and Mycobacterium tuberculosis ŽDifco, Detroit. at a final concentration of 0.5 mgrml. AT-EAN was induced by tail vein injection of 8 = 10 6 P2-specific CD4-positive activated T-cells. These neuritogenic T-cells were generated and maintained as previously described in detail ŽLinington et al., 1984..
2.6. Immunocytochemistry and histological analysis At the end of the experiment animals were anesthetized with Narcoren ŽIffa Merieux, Laupheim. at a dosage of 200 mgrkg body weight and perfused through the left
2.3. Treatment protocol Starting on the day of immunization or cell transfer six animals per group were treated with daily tail vein injec. at 400 mgrkg tions of Sandoglobinw ŽSandoz, Nurnberg ¨ body weight Žstandard dose treatment.. In a later experiment another group received 2000 mgrkg body weight Žhigh dose treatment. or sham treatment with PBS. One ml of IVIg or PBS were injected within one minute. Treatment was continued until day 4 after the maximum of disease or until the disease maximum Žhigh dose treatment. and animals were followed clinically for a further 6 days. In each experiment a pilot group of three animals was included which received IVIg only at the day of disease induction. 2.4. Determination of IVIg leÕels in rat sera The level of human IgG in rat sera was assessed with a Nephelometer Analyzer ŽBehring; Marburg.. To exclude rat anti-human antibodies sera were tested with a commercially available immunodiffusion assay ŽPartigen, Behringwerke.. 2.5. Scoring of disease Animals were weighed and inspected for disease severity daily by two investigators. Disease severity was assessed clinically employing a scale ranging from 0 to 10 ŽHartung et al., 1988; King et al., 1985.: 0 s normal, 1 s less lively, 2 s impaired rightingrlimb tail, 3 s absent righting, 4 s atactic gait, abnormal position, 5 s mild
Fig. 1. Disease course of active EAN ŽA. and mean body weight ŽB. in the different therapy groups. Treatment was started at day 0 in both groups and continued until day 16. The standard dose group received 400 mgrkg body weight, the high dose group 2000 mgrkg body weight. Time period of treatment is indicated at the bottom. Values are given as mean"SD Žstandard deviation. in groups of 6 animals each.
114
U. Enders et al.r Journal of Neuroimmunology 76 (1997) 112–116
cardiac ventricle with Ringer solution ŽFresenius, Bad Homburg. containing 2 = 10 4 Url heparin, followed by 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. For immunocytochemistry, 5 m m tissue sections were
stained with the monoclonal antibody ŽmAb. ED1 Ždilution 1:1000; Serotec, Wiesbaden. staining macrophages, mAb B115.1 Ždilution 1:500; Holland Biotechnology, Leiden. for T-cells and anti-human IgG serum ŽDako, Hamburg.
Fig. 2. Immunocytochemical detection of T-cells ŽA–B., macrophages ŽE–F., and human IgG ŽC–D. in sciatic nerve from control animals ŽA, C, and E. and the high dose treatment group ŽB, D and F.. Note that macrophages were stained in transverse sections to facilitate histological analysis. Bar s 100 m m.
U. Enders et al.r Journal of Neuroimmunology 76 (1997) 112–116
115
for staining human immunoglobulins deposited in peripheral nerves, as described by Heininger et al. Ž1984.. We then used the ABC detection system ŽDako, Hamburg. ´ with 3,3-diaminobenzidine as peroxidase substrate. Endogenous peroxidase activity was suppressed by incubating the sections for 10 min in 3% H 2 O 2 prior to the ABC reagent. Finally sections were counterstained with hematoxylin, dehydrated and mounted in Eukitt ŽKindler, Freiburg.. Coded sections were examined for density of infiltrating T-cells and macrophages by masked observers. 2.7. Statistical analysis One way Anova was performed for the comparison of body weight and T-cell density in inflamed peripheral nerves between both treatment groups and the control group. For the comparison of disease scores Kruskall– Wallis analysis was employed. All statistical tests were performed with GraphPad Prism w 2.0 ŽGraphPad Software, San Diego..
3. Results 3.1. ActiÕe EAN IVIg therapy was well tolerated. At day 10 after immunization with bovine peripheral myelin, animals in the IVIg and control groups suffered from weight loss and one day later they exhibited first clinical signs of EAN. Between days 15 to 18 all animals reached the peak of disease with severe paraparesis and slowly recovered thereafter. Neither for body weight nor for clinical score was there a statistically significant difference detected between the treatment and the placebo group Žclinical score at peak of disease: sham treated 5.5 " 1, IVIg treated 5.8 " 1.6; mean body weight at peak of disease: sham treated 138 " 15 g, IVIg treated 144 " 16 g.. Animals that received IVIg only at disease induction did also not benefit. In another experiment we investigated whether increasing the IVIg dose would exert a beneficial effect and injected animals with 2000 mg of IVIgrkg body weight as compared to a standard dose IVIg. Serum levels of human Ig were measured on days 10 and 15 after initiation of therapy ŽTable 1.. There was no difference in disease score between the treatment groups and control group Ždisease score P s 0.087; mean body weight P s 0.123; Fig. 1.. At the end of the experiment T-cell and macrophage inflammation of sciatic nerves was assessed by immunocytochemistry ŽFig. 2A–B, E–F.. Again there was no difference in T-cell density between the treatment groups ŽT-cell density: control group 128 " 42, standard dose group 163 " 52, high dose group 131 " 57 T-cells; P s 0.55.. Deposits of human IVIg were detected in sciatic nerves of IVIg recipients, but not in controls ŽFig. 2C–D..
Fig. 3. Disease course of AT-EAN ŽA. and mean body weight ŽB. in the therapy and the control group. Treatment was started with the standard dosage Ž400 mgrkg body weight. at day 0 and continued until day 7. Time period of treatment is indicated at the bottom. Values are given as mean"SD Žstandard deviation. in groups of 6 animals each.
Similar results were obtained in active EAN induced with the neuritogenic P2 peptide Ždata not shown.. 3.2. AT-EAN In this model the disease starts on day 3. At the maximum of disease animals in both groups exhibited severe tetraparesis ŽFig. 3.. No statistically significant difference was detected between the controls and the treated group.
4. Discussion The aim of this study was to establish that IVIg can modulateexperimental inflammatory demyelination in the PNS to obtain a model that would be suitable for the delineation of mechanisms underlying the therapeutic ef-
116
U. Enders et al.r Journal of Neuroimmunology 76 (1997) 112–116
fects of IVIg in GBS. Special emphasis was laid on the inflammatory phase of the disease, since IVIg has several potent anti-inflammatory effects Žreviewed in Stangel et al., 1997. and some of these mechanisms have been shown to be active in EAN Žreviewed in Hartung et al., 1995.. Unexpectedly the evaluation of IVIg treatment in different models of EAN has failed to demonstrate a salutary effect. This stands in contrast to the results in experimental autoimmune encephalomyelitis, the animal model for multiple sclerosis, where disease induced by active immunization could be suppressed with IVIg ŽAchiron et al., 1994.. There may be several reasons for the lack of a modulating impact of IVIg in EAN: theoretically human IVIg preparations could elicit neutralizing rat anti-human Ig. This was excluded by radial immunodiffusion assays where sera from control and IVIg treated rats were compared Ždata not shown.. In the study published by Achiron et al. Ž1994. the administration of IVIg resulted in a decreased level of TNFa secretion. This would argue for a modulation of the immune response after activation of autoreactive T-cells by recognition of self-antigens in EAE. Furthermore a single dose of IVIg at the time of immunization or cell transfer did not influence the clinical course of EAN Ždata not shown. whereas this strategy was effective as well in experimental autoimmune arthritis ŽAchiron et al., 1994.. While according to our results IVIg appears not to suppress the acute inflammatory insult on peripheral nerve, the possibility remains that it may have long term effects not formally looked for in the present observation i.e. facilitation of remyelination ŽRodriguez and Lennon, 1990.. Such investigations have recently been performed by Hughes and colleagues ŽGabriel et al., 1997.. These authors showed a modest benefit in the treatment group leading to accelerated recovery of the rats when looking at the post-inflammatory phase. It should be noted that the absolute differences between control and IVIg treated rats were small. It may be interesting to extend this particular effect of enhanced remyelination to chronic models of EAN, such as cyclosporin modulated EAN in the rat ŽMcCombe et al., 1990.. References Achiron, A., Margalit, R., Hershkoviz, R., Reshef, T., Melamed, E., Cohen, I.R. and Lider, O. Ž1994. Intravenous immunoglobulin treatment of experimental T-cell-mediated autoimmune disease. Upregulation of T-cell proliferation and downregulation of tumor necrosis factor alpha secretion. J. Clin. Invest. 93Ž2., 600–605.
Brostoff, S.W., Levit, S. and Powers, J.M. Ž1977. Induction ofexperimental allergic neuritis with a peptide from myelin P2 basic protein. Nature 268, 752–753. Gabriel, C.M., Gregson, N.A., Redford, E.J., Davies, M., Smith, K. and Hughes, R.A.C. Ž1997. Human immunoglobulin ameliorates rat experimental autoimmune neuritis ŽEAN.. Brain, in press. Hartung, H.-P., Pollard, J.D., Harvey, G.K. and Toyka, K.V. Ž1995. Immunopathogenesis and treatment of the Guillain-Barre-syndrome´ part II. Muscle Nerve 18, 154–164. Hartung, H.-P., Schafer, B., Heininger, K., Stoll, G. and Toyka, K.V. ¨ Ž1988. The role of macrophages and eicosanoids in the pathogenesis of experimental allergic neuritis. Brain 111, 1039–1059. Hartung, H.-P., Stoll, G., Toyka, K.V., Dyck, P.J., Thomas, P.K. and Griffin, J.W. ŽEds.. Ž1993. Immune reactions in the peripheral nervous system. In: Peripheral Neuropathy, Vol. 26. Saunders, Philadelphia, pp. 418–444. Heininger, K., Liebert, U.G., Toyka, K.V., Haneveld, F.F., Schwendemann, G., Kolb-Bachofen, V., Ross, H.G., Cleveland, S., Besinger, U.A. and Gibbels, E. Ž1984. Chronic inflammatory polyneuropathy. Reduction of nerve conduction velocities in monkeys by systemic passive transfer of immunoglobulins. J. Neurol. Sci. 66, 1–14. Kaveri, S.V., Dietrich, G. and Hurez, V. Ž1991. Intravenous immunoglobulins in the treatment of autoimmune disease. Clin. Exp. Immunol. 86, 192–198. King, R.H.M., Craggs, R.I., Cross, M.L.P. and Thomas, P.K. Ž1985. Effects of glucocorticoids on experimental allergic neuritis. Exp. Neurol. 87, 9–19. Linington, C., Izumo, S., Suzuki, M., Uyemura, K., Meyermann, R. and Wekerle, H. Ž1984. A permanent rat T-cell line that mediates experimental allergic neuritis in the Lewis rat in vivo. J. Immunol. 133, 1946–1950. McCombe, P.A., van der Kreek, S.A. and Pender, M.P. Ž1990. The effects of prophylactic cyclosporin A on experimental allergic neuritis ŽEAN. in the Lewis rat. Induction of relapsing EAN using low dose cyclosporin A. J. Neuroimmunol. 28, 131–140. Norton, W. and Poduslo, S. Ž1973. Myelination in rat brain-changes in myelin composition during brain maturation. J. Neurochem. 21, 759– 773. Plasma ExchangerSandoglobulin Guillain-Barre´ Trial Group Ž1997. Randomised trial of plasma exchange, intravenous immunoglobulin, and combined treatments in Guillain-Barre´ syndrome. Lancet 349, 225– 230. Rodriguez, M. and Lennon, V.A. Ž1990. Immunoglobulins promoteremyelination in the central nervous system. Ann. Neurol. 27, 7–12. Smith, M.E., Forno, L.S. and Hofmann, W.W. Ž1979. Experimental allergic neuritis in the Lewis rat. J. Neuropathol. Exp. Neurol. 38, 377–391. Stangel, M., Hartung, H.-P., Marx, P. and Gold, R. Ž1997. Intravenous immunoglobulin treatment of neurological autoimmune diseases. J. Neurol. Sci., in press. Uyemura, K., Tobari, C., Hirano, S. and Tsukada, Y. Ž1972. Comparative studies on the myelin proteins of bovine peripheral nerve and spinal cord. J. Neurochem. 19, 2607–2614. van der Meche, F.G., Schmitz, P.I. and the Dutch Guillain-Barre´ study group Ž1992. A randomized trial comparing intravenous immune globulin and plasma exchange in Guillain-Barre´ syndrome. N. Engl. J. Med. 326, 1123–1129.
Failure of intravenous immunoglobulin ž IVIg/ therapy in experimental autoimmune neuritis ž EAN/ of the Lewis rat U. Enders, K.V. Toyka, H.-P. Hartung, R. Gold
)
Department of Neurology, Neuroimmunology Branch and Clinical Research Group for Multiple Sclerosis, Julius-Maximilians UniÕersitat, ¨ Josef-Schneider-Straße 11, D-97080 Wurzburg, Germany ¨ Received 2 December 1996; revised 16 January 1997; accepted 17 January 1997
Abstract Experimental autoimmune neuritis ŽEAN. is an animal model for Guillain-Barre´ syndrome ŽGBS.. Intravenous immunoglobulins ŽIVIg. are an effective treatment for GBS, but their mechanism of action is not well understood. Here we tested whether IVIg treatment, a potent modulator of proinflammatory assaults, reduces inflammation in EAN. The evaluation of IVIg treatment failed to demonstrate a salutary effect in different models of EAN. IVIg appears not to suppress the acute inflammatory insult on the peripheral nerve, but may have beneficial long-term effects not looked for in the present investigation. Keywords: Immune globulin; Neuritis; Guillain-Barre´ syndrome
1. Introduction Intravenous administration of 7S-immunoglobulins ŽIVIg. obtained from pools of 10,000 or more donors is now an established therapy in immunopathologic disorders Žreviewed in Kaveri et al., 1991.. In the past decade the use of IVIg has been extended into the therapy of diverse neurological diseases thought to be autoimmune in nature. Recent reports Žvan der Meche et al., 1992. claimed a similar efficacy of high-dose IVIg and plasmapheresis in GBS, the most frequent cause of acute ascending paralysis. The results of the largest ever conducted trial in this disorder, comparing plasmapheresis ŽPE., IVIg and a combination of PE and IVIg, demonstrated equipotency of IVIg ŽPSGBS Group, 1997.. A multitude of potential mechanisms may underlie the beneficial actions of IVIg. Amongst these, inhibition of proinflammatory effects plays a central role. IVIg can act by anti-idiotypic effects on T-cell receptors, inhibition of complement-mediated cytotoxicity, modulation of cytokine production and direct binding of cytokines and blockade of
) Corresponding author. Tel.: q49-931-2012605; fax: q49-9312012697.
Fc receptors Žreviewed in Stangel et al., 1997.. Since systematic studies are not possible in patients we decided to investigate these actions in experimental autoimmune neuritis of Lewis rats, an animal model of GBS ŽHartung et al., 1993; Hartung et al., 1995.. We wished to address the effects of IgG on the different models of EAN and therefore used all three variants in the Lewis rat: EAN induced by active immunization with whole peripheral myelin ŽSmith et al., 1979., the neuritogenic peptide of the P2 protein ŽBrostoff et al., 1977., or by adoptive transfer of activated P2-specific neuritogenic T-lymphocytes ŽATEAN. ŽLinington et al., 1984.. We failed to demonstrate an immunomodulatory effect in either model.
2. Materials and methods 2.1. Animals Female Lewis rats were obtained from Charles River Laboratories ŽSulzfeld, Germany. and were 7–8 weeks old. Animals were housed in plastic cages without grid floors and given commercial food pellets and water ad libidum. All experiments were performed according to the Bavarian state regulations for animal experimentation and approved by the responsible authorities.
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 7 . 0 0 0 3 9 - 8
U. Enders et al.r Journal of Neuroimmunology 76 (1997) 112–116
113
Table 1 Human IgG in rat sera. Results are given as mgrdl Ž n s 6 rats per group. Day 10 Žmean " SD.
Day 15 Žmean " SD.
Controls Standard dose treatment Ž400 mgrkg body weight. High dose treatment Ž2000 mgrkg body weight.
73 " 15 2087 " 179 4736 " 562
145 " 41 1428 " 190 2532 " 161
2.2. Disease induction
paraparesis, 6 s moderate paraparesis, 7 s severe paraparesis, 8 s tetraparesis, 9 s moribund and 10 s death.
For active EAN, Lewis rats were inoculated into the footpad with 50 m l of an emulsion of an equal volume of saline and complete Freunds adjuvant containing 2 mg peripheral nerve myelin prepared from bovine spinal roots ŽNorton and Poduslo, 1973; Uyemura et al., 1972. or 100 m g P2 peptide spanning aa 53–78 of bovine P2 ŽMedprobe, Oslo. and Mycobacterium tuberculosis ŽDifco, Detroit. at a final concentration of 0.5 mgrml. AT-EAN was induced by tail vein injection of 8 = 10 6 P2-specific CD4-positive activated T-cells. These neuritogenic T-cells were generated and maintained as previously described in detail ŽLinington et al., 1984..
2.6. Immunocytochemistry and histological analysis At the end of the experiment animals were anesthetized with Narcoren ŽIffa Merieux, Laupheim. at a dosage of 200 mgrkg body weight and perfused through the left
2.3. Treatment protocol Starting on the day of immunization or cell transfer six animals per group were treated with daily tail vein injec. at 400 mgrkg tions of Sandoglobinw ŽSandoz, Nurnberg ¨ body weight Žstandard dose treatment.. In a later experiment another group received 2000 mgrkg body weight Žhigh dose treatment. or sham treatment with PBS. One ml of IVIg or PBS were injected within one minute. Treatment was continued until day 4 after the maximum of disease or until the disease maximum Žhigh dose treatment. and animals were followed clinically for a further 6 days. In each experiment a pilot group of three animals was included which received IVIg only at the day of disease induction. 2.4. Determination of IVIg leÕels in rat sera The level of human IgG in rat sera was assessed with a Nephelometer Analyzer ŽBehring; Marburg.. To exclude rat anti-human antibodies sera were tested with a commercially available immunodiffusion assay ŽPartigen, Behringwerke.. 2.5. Scoring of disease Animals were weighed and inspected for disease severity daily by two investigators. Disease severity was assessed clinically employing a scale ranging from 0 to 10 ŽHartung et al., 1988; King et al., 1985.: 0 s normal, 1 s less lively, 2 s impaired rightingrlimb tail, 3 s absent righting, 4 s atactic gait, abnormal position, 5 s mild
Fig. 1. Disease course of active EAN ŽA. and mean body weight ŽB. in the different therapy groups. Treatment was started at day 0 in both groups and continued until day 16. The standard dose group received 400 mgrkg body weight, the high dose group 2000 mgrkg body weight. Time period of treatment is indicated at the bottom. Values are given as mean"SD Žstandard deviation. in groups of 6 animals each.
114
U. Enders et al.r Journal of Neuroimmunology 76 (1997) 112–116
cardiac ventricle with Ringer solution ŽFresenius, Bad Homburg. containing 2 = 10 4 Url heparin, followed by 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. For immunocytochemistry, 5 m m tissue sections were
stained with the monoclonal antibody ŽmAb. ED1 Ždilution 1:1000; Serotec, Wiesbaden. staining macrophages, mAb B115.1 Ždilution 1:500; Holland Biotechnology, Leiden. for T-cells and anti-human IgG serum ŽDako, Hamburg.
Fig. 2. Immunocytochemical detection of T-cells ŽA–B., macrophages ŽE–F., and human IgG ŽC–D. in sciatic nerve from control animals ŽA, C, and E. and the high dose treatment group ŽB, D and F.. Note that macrophages were stained in transverse sections to facilitate histological analysis. Bar s 100 m m.
U. Enders et al.r Journal of Neuroimmunology 76 (1997) 112–116
115
for staining human immunoglobulins deposited in peripheral nerves, as described by Heininger et al. Ž1984.. We then used the ABC detection system ŽDako, Hamburg. ´ with 3,3-diaminobenzidine as peroxidase substrate. Endogenous peroxidase activity was suppressed by incubating the sections for 10 min in 3% H 2 O 2 prior to the ABC reagent. Finally sections were counterstained with hematoxylin, dehydrated and mounted in Eukitt ŽKindler, Freiburg.. Coded sections were examined for density of infiltrating T-cells and macrophages by masked observers. 2.7. Statistical analysis One way Anova was performed for the comparison of body weight and T-cell density in inflamed peripheral nerves between both treatment groups and the control group. For the comparison of disease scores Kruskall– Wallis analysis was employed. All statistical tests were performed with GraphPad Prism w 2.0 ŽGraphPad Software, San Diego..
3. Results 3.1. ActiÕe EAN IVIg therapy was well tolerated. At day 10 after immunization with bovine peripheral myelin, animals in the IVIg and control groups suffered from weight loss and one day later they exhibited first clinical signs of EAN. Between days 15 to 18 all animals reached the peak of disease with severe paraparesis and slowly recovered thereafter. Neither for body weight nor for clinical score was there a statistically significant difference detected between the treatment and the placebo group Žclinical score at peak of disease: sham treated 5.5 " 1, IVIg treated 5.8 " 1.6; mean body weight at peak of disease: sham treated 138 " 15 g, IVIg treated 144 " 16 g.. Animals that received IVIg only at disease induction did also not benefit. In another experiment we investigated whether increasing the IVIg dose would exert a beneficial effect and injected animals with 2000 mg of IVIgrkg body weight as compared to a standard dose IVIg. Serum levels of human Ig were measured on days 10 and 15 after initiation of therapy ŽTable 1.. There was no difference in disease score between the treatment groups and control group Ždisease score P s 0.087; mean body weight P s 0.123; Fig. 1.. At the end of the experiment T-cell and macrophage inflammation of sciatic nerves was assessed by immunocytochemistry ŽFig. 2A–B, E–F.. Again there was no difference in T-cell density between the treatment groups ŽT-cell density: control group 128 " 42, standard dose group 163 " 52, high dose group 131 " 57 T-cells; P s 0.55.. Deposits of human IVIg were detected in sciatic nerves of IVIg recipients, but not in controls ŽFig. 2C–D..
Fig. 3. Disease course of AT-EAN ŽA. and mean body weight ŽB. in the therapy and the control group. Treatment was started with the standard dosage Ž400 mgrkg body weight. at day 0 and continued until day 7. Time period of treatment is indicated at the bottom. Values are given as mean"SD Žstandard deviation. in groups of 6 animals each.
Similar results were obtained in active EAN induced with the neuritogenic P2 peptide Ždata not shown.. 3.2. AT-EAN In this model the disease starts on day 3. At the maximum of disease animals in both groups exhibited severe tetraparesis ŽFig. 3.. No statistically significant difference was detected between the controls and the treated group.
4. Discussion The aim of this study was to establish that IVIg can modulateexperimental inflammatory demyelination in the PNS to obtain a model that would be suitable for the delineation of mechanisms underlying the therapeutic ef-
116
U. Enders et al.r Journal of Neuroimmunology 76 (1997) 112–116
fects of IVIg in GBS. Special emphasis was laid on the inflammatory phase of the disease, since IVIg has several potent anti-inflammatory effects Žreviewed in Stangel et al., 1997. and some of these mechanisms have been shown to be active in EAN Žreviewed in Hartung et al., 1995.. Unexpectedly the evaluation of IVIg treatment in different models of EAN has failed to demonstrate a salutary effect. This stands in contrast to the results in experimental autoimmune encephalomyelitis, the animal model for multiple sclerosis, where disease induced by active immunization could be suppressed with IVIg ŽAchiron et al., 1994.. There may be several reasons for the lack of a modulating impact of IVIg in EAN: theoretically human IVIg preparations could elicit neutralizing rat anti-human Ig. This was excluded by radial immunodiffusion assays where sera from control and IVIg treated rats were compared Ždata not shown.. In the study published by Achiron et al. Ž1994. the administration of IVIg resulted in a decreased level of TNFa secretion. This would argue for a modulation of the immune response after activation of autoreactive T-cells by recognition of self-antigens in EAE. Furthermore a single dose of IVIg at the time of immunization or cell transfer did not influence the clinical course of EAN Ždata not shown. whereas this strategy was effective as well in experimental autoimmune arthritis ŽAchiron et al., 1994.. While according to our results IVIg appears not to suppress the acute inflammatory insult on peripheral nerve, the possibility remains that it may have long term effects not formally looked for in the present observation i.e. facilitation of remyelination ŽRodriguez and Lennon, 1990.. Such investigations have recently been performed by Hughes and colleagues ŽGabriel et al., 1997.. These authors showed a modest benefit in the treatment group leading to accelerated recovery of the rats when looking at the post-inflammatory phase. It should be noted that the absolute differences between control and IVIg treated rats were small. It may be interesting to extend this particular effect of enhanced remyelination to chronic models of EAN, such as cyclosporin modulated EAN in the rat ŽMcCombe et al., 1990.. References Achiron, A., Margalit, R., Hershkoviz, R., Reshef, T., Melamed, E., Cohen, I.R. and Lider, O. Ž1994. Intravenous immunoglobulin treatment of experimental T-cell-mediated autoimmune disease. Upregulation of T-cell proliferation and downregulation of tumor necrosis factor alpha secretion. J. Clin. Invest. 93Ž2., 600–605.
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