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Experimental autoimmune encephalomyelitis in mice with a targeted deletion of the inducible nitric oxide synthase gene: increased T-helper 1 response
Neuroscience Letters 358 (2004) 58–62 www.elsevier.com/locate/neulet
Experimental autoimmune encephalomyelitis in mice with a targeted deletion of the inducible nitric oxide synthase gene: increased T-helper 1 responseq Kai G. Kahla, Harald H.H.W. Schmidtb, Stefan Junga,1, Paula Shermanc, Klaus V. Toykaa,*, Ju¨rgen Zielaseka,* a
Department of Neurology, Julius-Maximilians-University, Josef-Schneider Str. 11, D-97080 Wu¨rzburg, Germany b The Rudolf-Buchheim-Institute for Pharmacology, Justus-Liebig-University, Giessen, Germany c University of North Carolina, Chapel Hill, NC, USA Received 26 September 2003; received in revised form 15 December 2003; accepted 15 December 2003
Abstract Mice with a targeted deletion of the cytokine-inducible nitric oxide synthase gene (iNOS2/2 ) show increased severity of experimental autoimmune encephalomyelitis (EAE). We studied the mechanisms of susceptibility to myelin-basic protein-induced ‘active’ EAE in iNOS2/2 mice. Spleen cells and lymph node cells from iNOS2/2 mice with EAE showed a significantly enhanced ex vivo proliferation and production of T-helper 1 (Th1) cytokines (interferon-g by 157 and 57% and tumor-necrosis-factor-alpha by 86 and 27%, respectively). We conclude that NO produced by iNOS plays a protective role in EAE probably by inhibiting the production of Th1 cytokines and T cell proliferation. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Inducible nitric oxide synthase knockout mice; Multiple sclerosis; T-helper 1/T-helper 2 immune response; Oxidative stress; Peroxynitrite
Several studies showed an activation of inducible nitric oxide synthase (iNOS) in the central nervous system (CNS) of multiple sclerosis patients [17]. Since the role of nitric oxide (NO) cannot be studied in multiple sclerosis, several groups utilized the animal model experimental autoimmune encephalomyelitis (EAE, [17]). While some studies showed a net detrimental effect of NO in EAE [17], we and others found aggravation of the disease in Lewis rats and DA rats after inhibition of iNOS [6,11,19]. Mice with a targeted deletion of the iNOS gene also had an increased EAE q Grant Information: This study was supported by a grant from the Bundesminister fu¨r Jugend, Familie und Gesundheit (Interdisciplinary Research Centre, Project Wu¨rzburg C-4) to KVT, JZ and HS, and institutional grants from the Julius-Maximilians University Wu¨rzburg. H.S. was additionally supported by the Thyssen foundation (1994/1), and the Sonderforschungsbereich SFB 547. * Corresponding authors. Department of Neurology, Julius-MaximiliansUniversity, Fu¨chsleinstrasse 15, D-97080 Wu¨rzburg, Germany. Tel.: þ 49931-2017-6000; fax: þ 49-931-2017-7550. E-mail address: [email protected] (J. Zielasek). 1 Present address: Department of Neurology, University of the Saarland, Homburg/Saar, Germany.
severity [5,12], but the underlying immune mechanism remained unclear. We therefore studied cytokine secretion, immune cell proliferation and NOS activities during EAE in a mouse strain with a targeted deletion of the iNOS gene [9]. We obtained iNOS2/2 mice and littermate controls from homozygous inbreeding of iNOS2/2 £ C57BL/6. EAE was induced with guinea pig myelin-basic protein (gpMBP, 4 mg/ml; [4]) and complete Freund’s adjuvant. A total of 200 ml Pertussis toxin (1 mg/ml; Sigma, Deisenhofen, Germany) was administered intraperitoneally on days 0 and 2 post immunization. EAE severity was checked daily using a fivegrade scoring system (cf. Table 1). For proliferation assays, lymph node cells (LNC) and spleen cells (SC) were prepared from inguinal and axillary lymph nodes and spleens from three iNOS2/2 and three iNOSþ/þ mice on day 10 after immunization with gpMBP. Cells were cultured in flat bottom 96-well microtiter plates in supplemented RPMI. The mitogen concanavalin A (ConA, Sigma; final concentration 2.5 mg/ml), the mouse T cell receptor alpha/beta-activating antibody H57 (30 mg/ml; [8]), purified protein derivative of Mycobacterium
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.12.095
K.G. Kahl et al. / Neuroscience Letters 358 (2004) 58–62
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Table 1 Induction of experimental autoimmune encephalomyelitis (EAE) in iNOS2/2 mice, littermate controls (iNOSþ/þ ), wildtype (wt) C57Bl6, and wildtype 129 Sv mice Mouse strain
iNOS2/2
iNOSþ/þ
C57/Bl6
129 SV
EAE incidence Day of disease onset Maximum disease score Severity of disease 5 (moribund or dead) 4 (tetraparesis) 3 (hindlimb paraplegia) 2 (paraparesis) 1 (tail paralysis)
9/10 18 ^ 3 4^2
9/13 16 ^ 3 2^2
3/10 21 ^ 3 1^0
1/8 21*
6 2 – – 1
1 – 5 2 1
– – – – 3
– – – – 1
Means ^ SD. *Only one animal showed mild signs of EAE.
tuberculosis (PPD; 10 mg/ml; Statens Serum Institute, Copenhagen, Denmark), or gpMBP (20 mg/ml) were added to 2.5 £ 105 LNC or 4 £ 105 SC per well. Control wells received pure RPMI. Cells were labeled during the second day of culture with tritiated thymidine (Amersham, Braunschweig, Germany) and were harvested 10 –12 h later. [3H]-thymidine incorporation was measured by liquid scintillation counting (Wallac 1205 Betaplate; Pharmacia). Data are given as mean counts per minute (CPM) of triplicate cultures. For the determination of cytokines, we generated culture supernatants from 2.5 £ 106 LNC or 4 £ 106 SC per 1 ml of restimulation medium in 24-well Nunclon dishes (Nunc, Roskilde, Denmark). Cells were activated with ConA (2.5 mg/ml) or H57 (30 mg/ml) or received pure RPMI as a control. Supernatants were harvested 48 h later and the concentrations of Th1 (IFNg, TNFa) and Th2 (IL-4, IL-10) cytokines were determined by ELISAs (Quantikinee, R&D Systems, Minneapolis, USA). For the detection of nitrotyrosine, Western blots were performed with supernatants of homogenates from snap frozen tissues using a mouse monoclonal antibody against nitrotyrosine [1]. Immunoreactive proteins were detected
Fig. 1. Proliferation of spleen cells and lymph node cells from iNOS2/2 and iNOSþ/þ mice. Spleen cells (panel A) and lymph node cells (panel B) were obtained 10 days after induction of experimental autoimmune encephalomyelitis, i.e. during the induction phase of the disease and before clinical symptoms occurred. Proliferation was assessed by measuring incorporation of tritiated thymidine (expressed as counts/minute) into nuclei of proliferating cells. Open bars: iNOS2/2 mice; filled bars: iNOSþ/þ mice. Activation with the mitogens concanavalin A (ConA), purified protein derivative (PPD) and guinea pig myelin basic protein (MBP) in parallel assays lead to a significantly higher proliferation of iNOS2/2 derived T cells as compared to cells from iNOSþ/þ littermate control mice.
Table 2 Cytokine production of lymph node cells and spleen cells of iNOS2/2 and iNOSþ/þ mice after EAE induction ConA iNOS2/2
ConA iNOSþ/þ
Lymph node cells IFNg TNFa IL-4 IL-10
15049 ^ 956** 1134 ^ 21* 33 ^ 10 1329 ^ 266
5844 ^ 1127 610 ^ 312 23 ^ 11 1236 ^ 188
Spleen cells IFNg TNFa IL-4 IL-10
20197 ^ 2896** 940 ^ 114* 11 ^ 11 1459 ^ 193
Cytokine (pg/ml)
Stimulus strain
12895 ^ 350 738 ^ 32 37 ^ 50 1529 ^ 144
H57 iNOS2/2
5599 ^ 362** 901 ^ 46 23 ^ 10 465 ^ 88
25895 ^ 2768 986 ^ 91 93 ^ 21 2292 ^ 103
H57 iNOSþ/þ
Unstimulated iNOS2/2
Unstimulated iNOSþ/þ
1661 ^ 549 866 ^ 250 10 ^ 12 427 ^ 114
584 ^ 61** 409 ^ 21 20 ^ 11 445 ^ 137
136 ^ 75 290 ^ 18 5^7 378 ^ 81
25627 ^ 1776 932 ^ 41 104 ^ 58 2205 ^ 167
288 ^ 158 234 ^ 14 n.d. 421 ^ 106
397 ^ 83 259 ^ 122 n.d. 473 ^ 113
Shown are means ^ SD. IL-4/IL-10, interleukin-4, interleukin-10; IFNg, interferon-gamma; and TNFa, tumor necrosis factor-alpha. *P , 0.01 iNOS2/2 vs. iNOSþ/þ . **P , 0.001 iNOS2/2 vs. iNOSþ/þ .
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with an enhanced chemiluminescence kit (Pierce Super Signal Blaze) and quantified by densitometry. Catalytic activities of NOS in tissue homogenates were assayed by the conversion of tritiated L -arginine to L -citrulline [10]. Tissue homogenates (50 ml) were incubated under Vmax conditions either with 1 mM CaCl2 and 50 nM calmodulin (for total NOS activity) or 1 mM EGTA (for calcium-independent iNOS/NOS2 activity). All samples were analyzed in triplicate. Statistical analyzes of EAE severity, cytokine production and cellular proliferation were performed using the twosided non-parametric Mann –Whitney U-test, and EAE incidences were compared with the two-sided Fisher’s exact test. Differences between means of the biochemical assays were analyzed by Student’s t-test (paired or unpaired). P , 0:05 was considered statistically significant. iNOS2/2 mice developed a significantly more severe disease with regard to the mean maximal clinical score when compared to all other strains tested (Table 1). While the disease incidence and mean day of disease onset were not significantly different between iNOS2/2 and iNOSþ/þ mice, iNOS2/2 mice reached significantly higher disease scores (Table 1). Some variability of EAE severity occurred as expected in groups of mice with different genetic background, e.g. wildtype mice and iNOS2/2 mice, but such differences could not explain the large differences between iNOS2/2 and iNOSþ/þ mice. In proliferation assays, we found a significantly increased spleen cell proliferation after stimulation with ConA, PPD and gpMBP in iNOS2/2 mice as compared to iNOSþ/þ mice (Fig. 1A). In unstimulated lymph node cells, spontaneous proliferation was very high as compared to unstimulated spleen cells from both strains of mice (Fig. 1B). No further enhancement of lymph node cell proliferation could be achieved with ConA, H57, PPD or gpMBP (Fig. 1B). However, mitogen treatment of lymph node cells led to a slight but statistically significant higher proliferation in iNOS2/2 mice as compared to iNOSþ/þ (Fig. 1B). Production of the Th1 cytokines IFNg and TNFa was enhanced by ConA and H57 in both lymph node cells and spleen cells from iNOS2/2 and iNOSþ/þ mice compared to unstimulated cells (Table 2). In both cell types, ConAinduced production of IFNg and TNFa was higher in iNOS2/2 mice than iNOSþ/þ mice (IFNg increased by 157% in lymph node cells and by 57% in spleen cells; TNFa increased by 86% in lymph node cells and by 27% in spleen cells; Table 2). Also, spontaneous production of IFNg was significantly enhanced in iNOS2/2 lymph node cells as compared to wildtype mice (Table 2), although the absolute levels were very low. Production of the Th2 cytokines IL-4 and IL-10 was induced by ConA and H57 in spleen cells of both strains of mice (Table 2). Lymph node cells produced only very small amounts of these Th2 cytokines, and even after stimulation with ConA or H57 only IL-10 but not IL-4 was significantly increased (Table 2). There were no
significant differences between iNOS2/2 and iNOSþ/þ mice in the production of IL-4 and IL-10. The role of cytokines in EAE was the subject of various studies but their putative role in iNOS2/2 mice EAE has not been studied before. We show an increased Th1 response and proliferation of spleen and lymph node cells from iNOS2/2 mice when they develop EAE. The stronger proliferation and Th1 cytokine secretion by iNOS2/2 splenocytes and lymph node cells was more pronounced or even only evident when lymphocytes were stimulated under experimental conditions providing a close physical contact with the appropriate antigen-presenting cells (APCs). Since APCs are the major source of NO we conclude that NO exerts its immunosuppressive action on
Fig. 2. Nitric oxide synthase (NOS) activities in the CNS. Brain stem (BST), cervical (CSC) and lumbar spinal cord (LSC), from iNOSþ/þ (wt) and iNOS22/2 (NOS22/2 ) mice under control conditions (OTX), or immunized with complete Freund’s adjuvant (CFA) only, and from mice with experimental autoimmune encephalomyelitis (EAE) (n ¼ 2 – 4), were homogenized and analyzed for NOS activity in the presence of calcium (all NOS) or presence of the calcium chelator EGTA (iNOS and calciumindependent cNOS activity) as described in text. Tissues are shown in rostro-caudal order. Data represent means ^ SEM of n ¼ 3 independent experiments. Note the different y-axis scales.
K.G. Kahl et al. / Neuroscience Letters 358 (2004) 58–62
lymphocytes mainly during the direct interaction between APCs and lymphocytes. Splenocytes from a different iNOS2/2 mouse strain also produced more IFNg and less IL-4 in an infection model [16], and NO was shown in vitro to inhibit the production of the proinflammatory cytokines IL-2 and IFNg [15]. These data indicate that NO suppresses the Th1 cytokine response, and that NO produced by iNOS is presumably the major factor in downregulating the autoimmune response to MBP in mice. NO may have additional anti-inflammatory effects [7,13, 18], and we show here that iNOS2/2 mice with EAE develop a shift of the immune response towards a Th1-like cytokine pattern. In wildtype and iNOS2/2 mice, iNOS induction as measured by calcium-independent NOS activity (Fig. 2) or as nitrotyrosine immunoreactivity (data not shown) could not be observed. This may be due to a too low detection limit for iNOS activity and iNOS immunoreactive protein in this model. The calcium-independent NOS activity in iNOS2/2 mice may be due to endothelial-type NOS, which may appear in a calcium-independent form as well [2]. NOS activity was reduced in the cervical and lumbar spinal cord of iNOS2/2 mice, where EAE is usually most pronounced. However, there was only a mild further decrease after EAE induction in the lumbar spinal cord (Fig. 2). Most of the NOS activity was calcium-dependent and the net NOS activity was somewhat higher in wild type mice than in iNOS2/2 mice, although we did not detect systematic differences due to CFA treatment, EAE induction or genotype (Fig. 2). We here for the first time looked at the role of all remaining NOS activity in the CNS once iNOS has been deleted. Since the remaining activity was largely calciumdependent, this may reflect the combined activities of NOS1 (neuronal NOS, nNOS) and NOS3 (endothelial NOS, eNOS). There was some residual calcium-independent NOS activity that may be attributed to NOS3 as has been previously described [2]. In accordance with our findings using nitrotyrosine immunoreactivity and NOS biochemical assays, we did not detect NOS2 (cytokine-inducible NOS) activity in wild type mice, which suggests that even in these mice NOS2 is not or only marginally upregulated when gpMBP or CFA are administered. The total NOS activity during EAE was much higher in wildtype than in iNOS2/2 mice (cf. Fig. 2, cervical and lumbar spinal cord), which suggests that the activity of NO producing enzymes other than iNOS, may play an active role in disease suppression. All NOS species could be operative at different anatomical sites. Taken together with our immunological data, our results suggest that the principal action may be in the immune system through peripheral T cell activation rather than locally in the CNS, although we cannot rule out that even the lower amounts of NOS activity in the cervical and spinal cord of iNOS2/2 mice played a local role in enhancing disease severity. The normal production of NO as shown in our iNOSþ/þ
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mice may confer resistance to EAE induction, similarly to PVG rats, in which EAE-resistance is conferred by an increased number of and NO production by splenic macrophages [3,14]. This is in accord with our cell culture results in iNOS2/2 mice. Thus, a picture emerges showing in three different rat strains and in iNOS2/2 mice that the immunologic effects of NO confer resistance against EAE. These findings caution against the use of NO synthase inhibitors in inflammatory CNS disorders such as multiple sclerosis before the complex role of NO is better understood.
Acknowledgements We thank Hiltrud Klu¨pfel, Gabi Ko¨llner, and Birgit Wagner for technical assistance. Supported by university research funds from the State of Bavaria.
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[12] U.C. Sahrbacher, F. Lechner, H.P. Eugster, K. Frei, H. Lassmann, A. Fontana, Mice with an inactivation of the inducible nitric oxide synthase gene are susceptible to experimental autoimmune encephalomyelitis, Eur. J. Immunol. 28 (1998) 1332–1338. [13] S.C. Sicher, G.W. Chung, M.A. Vazquez, C.Y. Lu, Augmentation or inhibition of IFN-gamma-induced MHC class II expression by lipopolysaccharides. The roles of TNF-alpha and nitric oxide, and the importance of the sequence of signalling, J. Immunol. 155 (1997) 5826–5834. [14] M.A. Staykova, W. Cowden, D.O. Willenborg, Macrophages and nitric oxide as the possible cellular and molecular basis for strain and gender differences in susceptibility to autoimmune central nervous system inflammation, Immunol. Cell Biol. 80 (2002) 188–197. [15] A.W. Taylor-Robinson, F.Y. Liew, A. Severn, D. Xu, S.J. McScorley, P. Garside, J. Padron, R.S. Phillips, Regulation of the immune
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Experimental autoimmune encephalomyelitis in mice with a targeted deletion of the inducible nitric oxide synthase gene: increased T-helper 1 responseq Kai G. Kahla, Harald H.H.W. Schmidtb, Stefan Junga,1, Paula Shermanc, Klaus V. Toykaa,*, Ju¨rgen Zielaseka,* a
Department of Neurology, Julius-Maximilians-University, Josef-Schneider Str. 11, D-97080 Wu¨rzburg, Germany b The Rudolf-Buchheim-Institute for Pharmacology, Justus-Liebig-University, Giessen, Germany c University of North Carolina, Chapel Hill, NC, USA Received 26 September 2003; received in revised form 15 December 2003; accepted 15 December 2003
Abstract Mice with a targeted deletion of the cytokine-inducible nitric oxide synthase gene (iNOS2/2 ) show increased severity of experimental autoimmune encephalomyelitis (EAE). We studied the mechanisms of susceptibility to myelin-basic protein-induced ‘active’ EAE in iNOS2/2 mice. Spleen cells and lymph node cells from iNOS2/2 mice with EAE showed a significantly enhanced ex vivo proliferation and production of T-helper 1 (Th1) cytokines (interferon-g by 157 and 57% and tumor-necrosis-factor-alpha by 86 and 27%, respectively). We conclude that NO produced by iNOS plays a protective role in EAE probably by inhibiting the production of Th1 cytokines and T cell proliferation. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Inducible nitric oxide synthase knockout mice; Multiple sclerosis; T-helper 1/T-helper 2 immune response; Oxidative stress; Peroxynitrite
Several studies showed an activation of inducible nitric oxide synthase (iNOS) in the central nervous system (CNS) of multiple sclerosis patients [17]. Since the role of nitric oxide (NO) cannot be studied in multiple sclerosis, several groups utilized the animal model experimental autoimmune encephalomyelitis (EAE, [17]). While some studies showed a net detrimental effect of NO in EAE [17], we and others found aggravation of the disease in Lewis rats and DA rats after inhibition of iNOS [6,11,19]. Mice with a targeted deletion of the iNOS gene also had an increased EAE q Grant Information: This study was supported by a grant from the Bundesminister fu¨r Jugend, Familie und Gesundheit (Interdisciplinary Research Centre, Project Wu¨rzburg C-4) to KVT, JZ and HS, and institutional grants from the Julius-Maximilians University Wu¨rzburg. H.S. was additionally supported by the Thyssen foundation (1994/1), and the Sonderforschungsbereich SFB 547. * Corresponding authors. Department of Neurology, Julius-MaximiliansUniversity, Fu¨chsleinstrasse 15, D-97080 Wu¨rzburg, Germany. Tel.: þ 49931-2017-6000; fax: þ 49-931-2017-7550. E-mail address: [email protected] (J. Zielasek). 1 Present address: Department of Neurology, University of the Saarland, Homburg/Saar, Germany.
severity [5,12], but the underlying immune mechanism remained unclear. We therefore studied cytokine secretion, immune cell proliferation and NOS activities during EAE in a mouse strain with a targeted deletion of the iNOS gene [9]. We obtained iNOS2/2 mice and littermate controls from homozygous inbreeding of iNOS2/2 £ C57BL/6. EAE was induced with guinea pig myelin-basic protein (gpMBP, 4 mg/ml; [4]) and complete Freund’s adjuvant. A total of 200 ml Pertussis toxin (1 mg/ml; Sigma, Deisenhofen, Germany) was administered intraperitoneally on days 0 and 2 post immunization. EAE severity was checked daily using a fivegrade scoring system (cf. Table 1). For proliferation assays, lymph node cells (LNC) and spleen cells (SC) were prepared from inguinal and axillary lymph nodes and spleens from three iNOS2/2 and three iNOSþ/þ mice on day 10 after immunization with gpMBP. Cells were cultured in flat bottom 96-well microtiter plates in supplemented RPMI. The mitogen concanavalin A (ConA, Sigma; final concentration 2.5 mg/ml), the mouse T cell receptor alpha/beta-activating antibody H57 (30 mg/ml; [8]), purified protein derivative of Mycobacterium
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.12.095
K.G. Kahl et al. / Neuroscience Letters 358 (2004) 58–62
59
Table 1 Induction of experimental autoimmune encephalomyelitis (EAE) in iNOS2/2 mice, littermate controls (iNOSþ/þ ), wildtype (wt) C57Bl6, and wildtype 129 Sv mice Mouse strain
iNOS2/2
iNOSþ/þ
C57/Bl6
129 SV
EAE incidence Day of disease onset Maximum disease score Severity of disease 5 (moribund or dead) 4 (tetraparesis) 3 (hindlimb paraplegia) 2 (paraparesis) 1 (tail paralysis)
9/10 18 ^ 3 4^2
9/13 16 ^ 3 2^2
3/10 21 ^ 3 1^0
1/8 21*
6 2 – – 1
1 – 5 2 1
– – – – 3
– – – – 1
Means ^ SD. *Only one animal showed mild signs of EAE.
tuberculosis (PPD; 10 mg/ml; Statens Serum Institute, Copenhagen, Denmark), or gpMBP (20 mg/ml) were added to 2.5 £ 105 LNC or 4 £ 105 SC per well. Control wells received pure RPMI. Cells were labeled during the second day of culture with tritiated thymidine (Amersham, Braunschweig, Germany) and were harvested 10 –12 h later. [3H]-thymidine incorporation was measured by liquid scintillation counting (Wallac 1205 Betaplate; Pharmacia). Data are given as mean counts per minute (CPM) of triplicate cultures. For the determination of cytokines, we generated culture supernatants from 2.5 £ 106 LNC or 4 £ 106 SC per 1 ml of restimulation medium in 24-well Nunclon dishes (Nunc, Roskilde, Denmark). Cells were activated with ConA (2.5 mg/ml) or H57 (30 mg/ml) or received pure RPMI as a control. Supernatants were harvested 48 h later and the concentrations of Th1 (IFNg, TNFa) and Th2 (IL-4, IL-10) cytokines were determined by ELISAs (Quantikinee, R&D Systems, Minneapolis, USA). For the detection of nitrotyrosine, Western blots were performed with supernatants of homogenates from snap frozen tissues using a mouse monoclonal antibody against nitrotyrosine [1]. Immunoreactive proteins were detected
Fig. 1. Proliferation of spleen cells and lymph node cells from iNOS2/2 and iNOSþ/þ mice. Spleen cells (panel A) and lymph node cells (panel B) were obtained 10 days after induction of experimental autoimmune encephalomyelitis, i.e. during the induction phase of the disease and before clinical symptoms occurred. Proliferation was assessed by measuring incorporation of tritiated thymidine (expressed as counts/minute) into nuclei of proliferating cells. Open bars: iNOS2/2 mice; filled bars: iNOSþ/þ mice. Activation with the mitogens concanavalin A (ConA), purified protein derivative (PPD) and guinea pig myelin basic protein (MBP) in parallel assays lead to a significantly higher proliferation of iNOS2/2 derived T cells as compared to cells from iNOSþ/þ littermate control mice.
Table 2 Cytokine production of lymph node cells and spleen cells of iNOS2/2 and iNOSþ/þ mice after EAE induction ConA iNOS2/2
ConA iNOSþ/þ
Lymph node cells IFNg TNFa IL-4 IL-10
15049 ^ 956** 1134 ^ 21* 33 ^ 10 1329 ^ 266
5844 ^ 1127 610 ^ 312 23 ^ 11 1236 ^ 188
Spleen cells IFNg TNFa IL-4 IL-10
20197 ^ 2896** 940 ^ 114* 11 ^ 11 1459 ^ 193
Cytokine (pg/ml)
Stimulus strain
12895 ^ 350 738 ^ 32 37 ^ 50 1529 ^ 144
H57 iNOS2/2
5599 ^ 362** 901 ^ 46 23 ^ 10 465 ^ 88
25895 ^ 2768 986 ^ 91 93 ^ 21 2292 ^ 103
H57 iNOSþ/þ
Unstimulated iNOS2/2
Unstimulated iNOSþ/þ
1661 ^ 549 866 ^ 250 10 ^ 12 427 ^ 114
584 ^ 61** 409 ^ 21 20 ^ 11 445 ^ 137
136 ^ 75 290 ^ 18 5^7 378 ^ 81
25627 ^ 1776 932 ^ 41 104 ^ 58 2205 ^ 167
288 ^ 158 234 ^ 14 n.d. 421 ^ 106
397 ^ 83 259 ^ 122 n.d. 473 ^ 113
Shown are means ^ SD. IL-4/IL-10, interleukin-4, interleukin-10; IFNg, interferon-gamma; and TNFa, tumor necrosis factor-alpha. *P , 0.01 iNOS2/2 vs. iNOSþ/þ . **P , 0.001 iNOS2/2 vs. iNOSþ/þ .
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with an enhanced chemiluminescence kit (Pierce Super Signal Blaze) and quantified by densitometry. Catalytic activities of NOS in tissue homogenates were assayed by the conversion of tritiated L -arginine to L -citrulline [10]. Tissue homogenates (50 ml) were incubated under Vmax conditions either with 1 mM CaCl2 and 50 nM calmodulin (for total NOS activity) or 1 mM EGTA (for calcium-independent iNOS/NOS2 activity). All samples were analyzed in triplicate. Statistical analyzes of EAE severity, cytokine production and cellular proliferation were performed using the twosided non-parametric Mann –Whitney U-test, and EAE incidences were compared with the two-sided Fisher’s exact test. Differences between means of the biochemical assays were analyzed by Student’s t-test (paired or unpaired). P , 0:05 was considered statistically significant. iNOS2/2 mice developed a significantly more severe disease with regard to the mean maximal clinical score when compared to all other strains tested (Table 1). While the disease incidence and mean day of disease onset were not significantly different between iNOS2/2 and iNOSþ/þ mice, iNOS2/2 mice reached significantly higher disease scores (Table 1). Some variability of EAE severity occurred as expected in groups of mice with different genetic background, e.g. wildtype mice and iNOS2/2 mice, but such differences could not explain the large differences between iNOS2/2 and iNOSþ/þ mice. In proliferation assays, we found a significantly increased spleen cell proliferation after stimulation with ConA, PPD and gpMBP in iNOS2/2 mice as compared to iNOSþ/þ mice (Fig. 1A). In unstimulated lymph node cells, spontaneous proliferation was very high as compared to unstimulated spleen cells from both strains of mice (Fig. 1B). No further enhancement of lymph node cell proliferation could be achieved with ConA, H57, PPD or gpMBP (Fig. 1B). However, mitogen treatment of lymph node cells led to a slight but statistically significant higher proliferation in iNOS2/2 mice as compared to iNOSþ/þ (Fig. 1B). Production of the Th1 cytokines IFNg and TNFa was enhanced by ConA and H57 in both lymph node cells and spleen cells from iNOS2/2 and iNOSþ/þ mice compared to unstimulated cells (Table 2). In both cell types, ConAinduced production of IFNg and TNFa was higher in iNOS2/2 mice than iNOSþ/þ mice (IFNg increased by 157% in lymph node cells and by 57% in spleen cells; TNFa increased by 86% in lymph node cells and by 27% in spleen cells; Table 2). Also, spontaneous production of IFNg was significantly enhanced in iNOS2/2 lymph node cells as compared to wildtype mice (Table 2), although the absolute levels were very low. Production of the Th2 cytokines IL-4 and IL-10 was induced by ConA and H57 in spleen cells of both strains of mice (Table 2). Lymph node cells produced only very small amounts of these Th2 cytokines, and even after stimulation with ConA or H57 only IL-10 but not IL-4 was significantly increased (Table 2). There were no
significant differences between iNOS2/2 and iNOSþ/þ mice in the production of IL-4 and IL-10. The role of cytokines in EAE was the subject of various studies but their putative role in iNOS2/2 mice EAE has not been studied before. We show an increased Th1 response and proliferation of spleen and lymph node cells from iNOS2/2 mice when they develop EAE. The stronger proliferation and Th1 cytokine secretion by iNOS2/2 splenocytes and lymph node cells was more pronounced or even only evident when lymphocytes were stimulated under experimental conditions providing a close physical contact with the appropriate antigen-presenting cells (APCs). Since APCs are the major source of NO we conclude that NO exerts its immunosuppressive action on
Fig. 2. Nitric oxide synthase (NOS) activities in the CNS. Brain stem (BST), cervical (CSC) and lumbar spinal cord (LSC), from iNOSþ/þ (wt) and iNOS22/2 (NOS22/2 ) mice under control conditions (OTX), or immunized with complete Freund’s adjuvant (CFA) only, and from mice with experimental autoimmune encephalomyelitis (EAE) (n ¼ 2 – 4), were homogenized and analyzed for NOS activity in the presence of calcium (all NOS) or presence of the calcium chelator EGTA (iNOS and calciumindependent cNOS activity) as described in text. Tissues are shown in rostro-caudal order. Data represent means ^ SEM of n ¼ 3 independent experiments. Note the different y-axis scales.
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lymphocytes mainly during the direct interaction between APCs and lymphocytes. Splenocytes from a different iNOS2/2 mouse strain also produced more IFNg and less IL-4 in an infection model [16], and NO was shown in vitro to inhibit the production of the proinflammatory cytokines IL-2 and IFNg [15]. These data indicate that NO suppresses the Th1 cytokine response, and that NO produced by iNOS is presumably the major factor in downregulating the autoimmune response to MBP in mice. NO may have additional anti-inflammatory effects [7,13, 18], and we show here that iNOS2/2 mice with EAE develop a shift of the immune response towards a Th1-like cytokine pattern. In wildtype and iNOS2/2 mice, iNOS induction as measured by calcium-independent NOS activity (Fig. 2) or as nitrotyrosine immunoreactivity (data not shown) could not be observed. This may be due to a too low detection limit for iNOS activity and iNOS immunoreactive protein in this model. The calcium-independent NOS activity in iNOS2/2 mice may be due to endothelial-type NOS, which may appear in a calcium-independent form as well [2]. NOS activity was reduced in the cervical and lumbar spinal cord of iNOS2/2 mice, where EAE is usually most pronounced. However, there was only a mild further decrease after EAE induction in the lumbar spinal cord (Fig. 2). Most of the NOS activity was calcium-dependent and the net NOS activity was somewhat higher in wild type mice than in iNOS2/2 mice, although we did not detect systematic differences due to CFA treatment, EAE induction or genotype (Fig. 2). We here for the first time looked at the role of all remaining NOS activity in the CNS once iNOS has been deleted. Since the remaining activity was largely calciumdependent, this may reflect the combined activities of NOS1 (neuronal NOS, nNOS) and NOS3 (endothelial NOS, eNOS). There was some residual calcium-independent NOS activity that may be attributed to NOS3 as has been previously described [2]. In accordance with our findings using nitrotyrosine immunoreactivity and NOS biochemical assays, we did not detect NOS2 (cytokine-inducible NOS) activity in wild type mice, which suggests that even in these mice NOS2 is not or only marginally upregulated when gpMBP or CFA are administered. The total NOS activity during EAE was much higher in wildtype than in iNOS2/2 mice (cf. Fig. 2, cervical and lumbar spinal cord), which suggests that the activity of NO producing enzymes other than iNOS, may play an active role in disease suppression. All NOS species could be operative at different anatomical sites. Taken together with our immunological data, our results suggest that the principal action may be in the immune system through peripheral T cell activation rather than locally in the CNS, although we cannot rule out that even the lower amounts of NOS activity in the cervical and spinal cord of iNOS2/2 mice played a local role in enhancing disease severity. The normal production of NO as shown in our iNOSþ/þ
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mice may confer resistance to EAE induction, similarly to PVG rats, in which EAE-resistance is conferred by an increased number of and NO production by splenic macrophages [3,14]. This is in accord with our cell culture results in iNOS2/2 mice. Thus, a picture emerges showing in three different rat strains and in iNOS2/2 mice that the immunologic effects of NO confer resistance against EAE. These findings caution against the use of NO synthase inhibitors in inflammatory CNS disorders such as multiple sclerosis before the complex role of NO is better understood.
Acknowledgements We thank Hiltrud Klu¨pfel, Gabi Ko¨llner, and Birgit Wagner for technical assistance. Supported by university research funds from the State of Bavaria.
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