- Email: [email protected]
The role of reactive oxygen species in the phagocytosis of myelin
Poster Abstracts /Journal of Neuroimmunology 90 (1998) 13-105
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Synergistic Induction of TNF-~ Expression by
~x[3 (1-42) a n d I F N - 7 in Microgllal Cells is I n h i b i t e d by IL-4,1L-10 and IL-13 A.M, Szczepanik. W.W. Petko, G.E. Ringheim, HoechstMarionRoussel, Inc., NJ, USA
T h e Role of Reactive Oxygen Species in t h e Phagoeytosis o f Myelin A. van der Goes. K. Hoekstra, J. Brouwer, Frije Universiteit, Dept. CellBiology, The Netherlands, D. Roos, CLB,Dept. Exper~ental Immun. HaematoL, The Netherlands, T.K. van den Berg, C.D. Dijkstra, Vrije Universiteit, Dept. CellBiology, The Netherlands
Beta amyloid and IFN-y are known to synergistically activate microglia to re ~ase proinflammatory cytokines such as TNF-cc We investigated whether it,-4, IL-10, and IL-t 3 - cytokines with down modulating effects on certain microgliaYmacrophage functions - could also regulate TNF-a expression induced by beta amyloid and IFN-y. In primary conical microgliai cells, I00 pM AI3(1-42) in the presence of 10 U/ml IFN-~¢ was shown to induce maximal levels of secreted TNF-a by 24 h in a dose-responsive manner, with TNF-ct expression returning to basal levels by 48 h. Similar results were observed in the microglial cell line MG7 except that secretion of TNF~x remained maximal at 48h. The cytokiues IL-4, IL-10 and IL-13 were found to dose-dependently reduce TNF-ct levels induced by either AI3(I42)/IFN-y co-treatment or by lipopolysaccharide (LPS). While the interleukins appeared equally effective at inhibiting TNF-~x levels induced by A[3(1-42)/IFN-~' in primary microglial cells, IL-10 appeared to by the most effective at inhibiting TNF-cx expression induced by LPS~
Reactive oxygen species (ROS) are thought to be involved in the pathogenesis of multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). Not only are they able of damaging the lipid bilayer of surrounding ceils, such as endothelial cell of the blood-brain barrier and oligodendrceytes, but they can also damage the myelin sheath. In this study we show that ROS are involved in the phagocytosis of myelin by macrophages. We used an in vitro myelin phagocytosis assay. Fluorescent labeled myelin was feaded to macrophages in presence and absence of various ROS scavengers. Further, ROS production by macrophages after myelin phagocylosis was determined. The results showed that ROS are produced by macrophages during the phagocytosis of myelin. Scavenging of ROS with eatalase or manultol decreased the phagocytosis of myelin by macrophages, while superoxide dismutase did not show this effect. Lipoic acid a non-specific scavenger of ROS could also decrease the phagocytosis of myelin by macrophages. From our results, we conclude that ROS appear to play a regulatory role in the phagocytosis of myelin.
288
291
Characteriz ation of Pro- and A n t i - i n f l a m m a t o r y
Interleukin-6 Regulation in Human Astrocytes: Synergy Between TNF-alpha, Interleukin-6, and the SolubleInterleukin-6 Receptor-alpha
Cytokines in the C e n t r a l N e r v o u s S y s t e m in Two T y p e s of C h r o n i c R e l a p s i n g A u t o i m m u n e Encephalomyelitis N. Tanuma. TokyoMetropolitan Medical Centerfor the SeverelyHandicapped, Japan, G. Kim, K. Kohyam a, Y. Mats umoto, TokyoMetropolitan lnstiaaefor Neuroscience, Japan To elucidate the mechanisms of relapses of the clinical signs in expenmeatal autoimmune encephalomyelitis 0EAE), we induced chronic relapsing EAE in Lewis rats with cyclosporin (CsA-EAE) and DA mrs (DA-EAE) and determined the cytokine profile in the centnd nervous system by competitive PCR. The expression of TNF-ctmRNA was elevated during the first and second attacks of CsA-EAE and DA-EAE suggesting that the level of TNF-ct paralleled the clinical signs of EAE. The level of IFN-ymRNA was suppressed at the first attack and peaked at the second attack in CsA-EAE~ whereas it was elevated at the first and second attacks in DA-EAE. In addition, an intraventricular injection of IFN-Tprior to onset of CsA-EAE induced more relapses or a chronic persistent course. These findings suggest that IFN-"/is not required for the dise2.se initiation but is closely related to the disease progression. With regard to antiinflammatory cytokines, beth TGF-131 and IL-10 mRNA were detected at all time points of the disease in CsA-EAE. In contrast, in DA-EAE, the expression of these cytokines was low during the first attack and was elevated at the second attack. Taken together, the clinical course of CsA-EAE is differentially regulatedby TNF-ff.and IFN-~ On the other hand. the low levels of anti-inflammatory cytokines at the first attack may lead to the clinical relapse in DA-EAE.
N.J. Van Wagoner. E.N. Benveniste, University ofAlabama atBirmingharn, USA
[nterleukin-6 (IL-6) has both neurotrophic and immunologic functions, In the central nervous system (CNS), astrocytes are a major inducible source of IL-6, Specifically, the cytokine TNF-cz upregulates IL-6 expression in astrocytes. We address IL-6 autoregulation in human astroglionm cells and primary asuocytes to determine if IL-6 has an effect alone or in combination with TNF-cz on IL-6 regulation. [L-6 ~tlone or IL-6 plus the soluble IL-6 receptor-~, (sIL-6R~x) had no effect on IL-6 inc[uction. High levels of IL-6 protein and mRNA were induced with [L-6/slL-6R~x plus TNFqx. As well, TNF-ot synergized with slL-6Rct alone for enhanced IL-6 production, suggesting that endogenously produced IL-6 (induced by TNF-o0 complexed with slL-6Rtx lor enhanced IL-6 expression. This synergy was blocked using neutralizing antibodies to gpl30, the slL-6Ro~, and Ii,-6. Results indicate that IL-6, when provided with the slL-6Rct to initiate signaling, enhances TNF-ct mediated IL-6 production. Since IL-6 has been shown to downregulate TNF-~x induced responses, this upregulation of IL-6 in the presence of TNF-c~ and IL-6/slL-6R~x may serve as a mechanism for IL-6 mediated downregnlation of "I'NF-o. induced responses. Amflysis is underway to determine if IL-6, slL-6Ro~, and TNF-c~ coordinately upregulate the IL-6 receptors gpl30 and IL-6R~x in astrocytes to increase IL-6 responsiveness.
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Signal Regulatory Protein (SIRP) is Selectively Expressed by M a e r o p h a g e s a n d N e u r o n s a n d Mediates MacrophageAcfivafion
The C o m p l e m e n t A n a p h y l a t o x i n
T.K. van den Ber~. A. van der Goes, C.R. Delavalette, Vrije University, Amsterdam, The Netherlands, S. Adams, D.L. Simmons, lnstitute forMoLMedicine, Oxford, UK, C.D. Dijkstra, V'rijeUniversity, Art~terdam, TheNetherlands
Activated macrophages/mieroglial ceils (mq)) are the major cause of tissue damage during inflammation in the central nervous system (CNS) However, the actual mechanisms of mq~ activation are not exactly known. Here we demonstrate by expression cloning that the monoclonal antibodies ED9, EDI7 and MRC-OX41 recognize rat signal regulatory protein (SIRP) SIRPs are transmembrane glycoproteins with 3 extracel/ular lglike domains, closely related to antigen receptors Ig, TCR and MHC, and a cytoplasmic domain with two "immunoreceptor with tyrosine-based inhibition motifs" (ITIMs), that can interact with SH2-domain-containiug phosphatases. We also show for the first time that rat SIRP is selectively expressed by myeloid cells (mo, monocytes, granutocytes, dendritic cells) and neurons. SIRP mediates the binding and/or phagocytosis of myelin by into in vitro and SIRP ligation induces rag, activation (production of reactive oxygen species, nitric oxide, cytokines and chemokines) It is concluded that: [) SIRP is a putative recognition/signalling receptor in both immune and nervous systems and 2) SIRP may play a critical role in ml~ activation during CNS inflammation.
CSa Stimulates IL-8 Secretion b y Astroeytes A. Zhaldlov, Res. institute of Pure Biochemicals, Russia, S. Sayah, M. Fontaine, 1FRM~23, INSERM U78, France~ A. Isuhenko, Res. lnstiOae of Pure Biochemieal~; Russi~
Astrocytes produce "in vitro" a complete functional complement system, and wben activated, a set of cytokincs. They also express constitutiveh,' complement receptors CR2 and C3aR. C5aR. It may be speculated thfft complement activated products, C3a and CSa anaphylatoxins, may induce an army of functional responses by astr.oc2(tes via specific interactions with their receptors. To investigate this possib!l'!ty we have studied the induction of synthesis and secretion of the chemokine IL8 by human astrocyte cell lines with purified C3a and C5a and compared their effects to other chemotactic agents (fMLP and Substance P (SP)), as well as LPS. Secretion of IL8 was evaluated by a l'dghly specific ELISA (20 pg/ml). Our results clearly demonstrated that among the inducers studied, onl-y C5a was able to stimulate IL8 secretion by astrocvtes, The effect was dose-dependent in the range of concentration studied (0.5-5nm). IL8 concentration in cell supernatants increased with the time of i~ubation and reached a maxinarm at 48h witb a secretion rate of 10ng/ml/10 ° cells. C5adesarg possessed lesser potency to stimulate the chemokme secretion than C5a. C3a, as well as IMLP, SP and LPS were not able to induce IL8 secretion by astrocytic cells even at higher concentrations. Co-incubation with LPS weakly ntodulated dynamics of C5a-mediated IL-8 secretion. By contrast. C3a enhanced effect or C5a induced IL8 secretion. This potentmlingeffect may be due to a "priming" of astrocytes by C3a. The mechanism~ov which CSa induced IL8 secretion is not known so far. It may act either difecflv or via generation of active substances like reactive oxygen species or other ~ytokines.
53
287
290
Synergistic Induction of TNF-~ Expression by
~x[3 (1-42) a n d I F N - 7 in Microgllal Cells is I n h i b i t e d by IL-4,1L-10 and IL-13 A.M, Szczepanik. W.W. Petko, G.E. Ringheim, HoechstMarionRoussel, Inc., NJ, USA
T h e Role of Reactive Oxygen Species in t h e Phagoeytosis o f Myelin A. van der Goes. K. Hoekstra, J. Brouwer, Frije Universiteit, Dept. CellBiology, The Netherlands, D. Roos, CLB,Dept. Exper~ental Immun. HaematoL, The Netherlands, T.K. van den Berg, C.D. Dijkstra, Vrije Universiteit, Dept. CellBiology, The Netherlands
Beta amyloid and IFN-y are known to synergistically activate microglia to re ~ase proinflammatory cytokines such as TNF-cc We investigated whether it,-4, IL-10, and IL-t 3 - cytokines with down modulating effects on certain microgliaYmacrophage functions - could also regulate TNF-a expression induced by beta amyloid and IFN-y. In primary conical microgliai cells, I00 pM AI3(1-42) in the presence of 10 U/ml IFN-~¢ was shown to induce maximal levels of secreted TNF-a by 24 h in a dose-responsive manner, with TNF-ct expression returning to basal levels by 48 h. Similar results were observed in the microglial cell line MG7 except that secretion of TNF~x remained maximal at 48h. The cytokiues IL-4, IL-10 and IL-13 were found to dose-dependently reduce TNF-ct levels induced by either AI3(I42)/IFN-y co-treatment or by lipopolysaccharide (LPS). While the interleukins appeared equally effective at inhibiting TNF-~x levels induced by A[3(1-42)/IFN-~' in primary microglial cells, IL-10 appeared to by the most effective at inhibiting TNF-cx expression induced by LPS~
Reactive oxygen species (ROS) are thought to be involved in the pathogenesis of multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). Not only are they able of damaging the lipid bilayer of surrounding ceils, such as endothelial cell of the blood-brain barrier and oligodendrceytes, but they can also damage the myelin sheath. In this study we show that ROS are involved in the phagocytosis of myelin by macrophages. We used an in vitro myelin phagocytosis assay. Fluorescent labeled myelin was feaded to macrophages in presence and absence of various ROS scavengers. Further, ROS production by macrophages after myelin phagocylosis was determined. The results showed that ROS are produced by macrophages during the phagocytosis of myelin. Scavenging of ROS with eatalase or manultol decreased the phagocytosis of myelin by macrophages, while superoxide dismutase did not show this effect. Lipoic acid a non-specific scavenger of ROS could also decrease the phagocytosis of myelin by macrophages. From our results, we conclude that ROS appear to play a regulatory role in the phagocytosis of myelin.
288
291
Characteriz ation of Pro- and A n t i - i n f l a m m a t o r y
Interleukin-6 Regulation in Human Astrocytes: Synergy Between TNF-alpha, Interleukin-6, and the SolubleInterleukin-6 Receptor-alpha
Cytokines in the C e n t r a l N e r v o u s S y s t e m in Two T y p e s of C h r o n i c R e l a p s i n g A u t o i m m u n e Encephalomyelitis N. Tanuma. TokyoMetropolitan Medical Centerfor the SeverelyHandicapped, Japan, G. Kim, K. Kohyam a, Y. Mats umoto, TokyoMetropolitan lnstiaaefor Neuroscience, Japan To elucidate the mechanisms of relapses of the clinical signs in expenmeatal autoimmune encephalomyelitis 0EAE), we induced chronic relapsing EAE in Lewis rats with cyclosporin (CsA-EAE) and DA mrs (DA-EAE) and determined the cytokine profile in the centnd nervous system by competitive PCR. The expression of TNF-ctmRNA was elevated during the first and second attacks of CsA-EAE and DA-EAE suggesting that the level of TNF-ct paralleled the clinical signs of EAE. The level of IFN-ymRNA was suppressed at the first attack and peaked at the second attack in CsA-EAE~ whereas it was elevated at the first and second attacks in DA-EAE. In addition, an intraventricular injection of IFN-Tprior to onset of CsA-EAE induced more relapses or a chronic persistent course. These findings suggest that IFN-"/is not required for the dise2.se initiation but is closely related to the disease progression. With regard to antiinflammatory cytokines, beth TGF-131 and IL-10 mRNA were detected at all time points of the disease in CsA-EAE. In contrast, in DA-EAE, the expression of these cytokines was low during the first attack and was elevated at the second attack. Taken together, the clinical course of CsA-EAE is differentially regulatedby TNF-ff.and IFN-~ On the other hand. the low levels of anti-inflammatory cytokines at the first attack may lead to the clinical relapse in DA-EAE.
N.J. Van Wagoner. E.N. Benveniste, University ofAlabama atBirmingharn, USA
[nterleukin-6 (IL-6) has both neurotrophic and immunologic functions, In the central nervous system (CNS), astrocytes are a major inducible source of IL-6, Specifically, the cytokine TNF-cz upregulates IL-6 expression in astrocytes. We address IL-6 autoregulation in human astroglionm cells and primary asuocytes to determine if IL-6 has an effect alone or in combination with TNF-cz on IL-6 regulation. [L-6 ~tlone or IL-6 plus the soluble IL-6 receptor-~, (sIL-6R~x) had no effect on IL-6 inc[uction. High levels of IL-6 protein and mRNA were induced with [L-6/slL-6R~x plus TNFqx. As well, TNF-ot synergized with slL-6Rct alone for enhanced IL-6 production, suggesting that endogenously produced IL-6 (induced by TNF-o0 complexed with slL-6Rtx lor enhanced IL-6 expression. This synergy was blocked using neutralizing antibodies to gpl30, the slL-6Ro~, and Ii,-6. Results indicate that IL-6, when provided with the slL-6Rct to initiate signaling, enhances TNF-ct mediated IL-6 production. Since IL-6 has been shown to downregulate TNF-~x induced responses, this upregulation of IL-6 in the presence of TNF-c~ and IL-6/slL-6R~x may serve as a mechanism for IL-6 mediated downregnlation of "I'NF-o. induced responses. Amflysis is underway to determine if IL-6, slL-6Ro~, and TNF-c~ coordinately upregulate the IL-6 receptors gpl30 and IL-6R~x in astrocytes to increase IL-6 responsiveness.
289
292
Signal Regulatory Protein (SIRP) is Selectively Expressed by M a e r o p h a g e s a n d N e u r o n s a n d Mediates MacrophageAcfivafion
The C o m p l e m e n t A n a p h y l a t o x i n
T.K. van den Ber~. A. van der Goes, C.R. Delavalette, Vrije University, Amsterdam, The Netherlands, S. Adams, D.L. Simmons, lnstitute forMoLMedicine, Oxford, UK, C.D. Dijkstra, V'rijeUniversity, Art~terdam, TheNetherlands
Activated macrophages/mieroglial ceils (mq)) are the major cause of tissue damage during inflammation in the central nervous system (CNS) However, the actual mechanisms of mq~ activation are not exactly known. Here we demonstrate by expression cloning that the monoclonal antibodies ED9, EDI7 and MRC-OX41 recognize rat signal regulatory protein (SIRP) SIRPs are transmembrane glycoproteins with 3 extracel/ular lglike domains, closely related to antigen receptors Ig, TCR and MHC, and a cytoplasmic domain with two "immunoreceptor with tyrosine-based inhibition motifs" (ITIMs), that can interact with SH2-domain-containiug phosphatases. We also show for the first time that rat SIRP is selectively expressed by myeloid cells (mo, monocytes, granutocytes, dendritic cells) and neurons. SIRP mediates the binding and/or phagocytosis of myelin by into in vitro and SIRP ligation induces rag, activation (production of reactive oxygen species, nitric oxide, cytokines and chemokines) It is concluded that: [) SIRP is a putative recognition/signalling receptor in both immune and nervous systems and 2) SIRP may play a critical role in ml~ activation during CNS inflammation.
CSa Stimulates IL-8 Secretion b y Astroeytes A. Zhaldlov, Res. institute of Pure Biochemicals, Russia, S. Sayah, M. Fontaine, 1FRM~23, INSERM U78, France~ A. Isuhenko, Res. lnstiOae of Pure Biochemieal~; Russi~
Astrocytes produce "in vitro" a complete functional complement system, and wben activated, a set of cytokincs. They also express constitutiveh,' complement receptors CR2 and C3aR. C5aR. It may be speculated thfft complement activated products, C3a and CSa anaphylatoxins, may induce an army of functional responses by astr.oc2(tes via specific interactions with their receptors. To investigate this possib!l'!ty we have studied the induction of synthesis and secretion of the chemokine IL8 by human astrocyte cell lines with purified C3a and C5a and compared their effects to other chemotactic agents (fMLP and Substance P (SP)), as well as LPS. Secretion of IL8 was evaluated by a l'dghly specific ELISA (20 pg/ml). Our results clearly demonstrated that among the inducers studied, onl-y C5a was able to stimulate IL8 secretion by astrocvtes, The effect was dose-dependent in the range of concentration studied (0.5-5nm). IL8 concentration in cell supernatants increased with the time of i~ubation and reached a maxinarm at 48h witb a secretion rate of 10ng/ml/10 ° cells. C5adesarg possessed lesser potency to stimulate the chemokme secretion than C5a. C3a, as well as IMLP, SP and LPS were not able to induce IL8 secretion by astrocytic cells even at higher concentrations. Co-incubation with LPS weakly ntodulated dynamics of C5a-mediated IL-8 secretion. By contrast. C3a enhanced effect or C5a induced IL8 secretion. This potentmlingeffect may be due to a "priming" of astrocytes by C3a. The mechanism~ov which CSa induced IL8 secretion is not known so far. It may act either difecflv or via generation of active substances like reactive oxygen species or other ~ytokines.