ADONIS 000991049100045L
Clin. exp. Immunol. (1991) 83, 245-250
Antibody-mediated demyelination in experimental allergic encephalomyelitis is independent of complement membrane attack complex formation S. PIDDLESDEN, H. LASSMANNt, I. LAFFAFIAN*, B. P. MORGAN* & C. LININGTON: Section of Neurology, Department of Medicine, and *Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, Wales, tInstitute of Brain Research, Austrian Academy of Science, Vienna, Austria, and tDepartment of Neuroimmunology, Max-Planck Institute for Psychiatry, Munich, Germany
(Acceptedfor publication 21 September 1990)
SUMMARY The effects of decomplementation by cobra venom factor (CVF) on the pathogenesis of inflammation and demyelination in experimental allergic encephalomyelitis (EAE) and acute antibody-mediated demyelinating EAE (ADEAE) have been quantified histologically and immunocytochemically. In rats immunized with 50 yg of myelin basic protein in Freund's complete adjuvant containing 100 yug heat-killed Mycobacterium tuberculosis H37Ra, clinical signs of EAE were completely suppressed by two injections of CVF given 9 and 12 days post-immunization. Suppression of clinical disease was associated with a dramatic reduction in peri-vascular inflammation in the CNS, although immunohistochemical staining identified small numbers of infiltrating T cells and macrophages. In contrast, CVF treatment had no significant effect on the clinical severity of ADEAE and although C9 deposition within the CNS was virtually abolished, there was no statistically significant decrease in the extent of demyelination or inflammation. These observations indicate that in the absence of complement components C3 and C5 an antibody-dependent cell-mediated cytotoxic response plays an important role in the pathogenesis of antibody-mediated demyelination. The major role of the complement cascade in EAE appears to be the generation of pro-inflammatory factors that enhance the inflammatory response within the CNS in animals facing a mild encephalitogenic challenge.
Keywords
demyelination complement inflammation experimental allergic encephalomyelitis
anti-myelin antibody subsequently initiates demyelination is unclear. MoAb 8-18C5 is of the IgGI subclass and is capable of complement fixation (Linington et al., 1989b). Complementdependent mechanisms involving complement activation by the 8-18C5 MoAb and membrane attack complex (MAC) formation have been shown to be cytolytic to MOG+ oligodendrocytes in vitro (Linington et al., 1 989b), and the antibody initiates extensive C9 deposition in the white matter tracts of animals with ADEAE (Linington et al., 1989a). However, other studies have indicated that antibody-mediated demyelination in vivo involves an antibody-dependent cellular cytotoxicity (ADCC) mechanism (Brosnan, Bornstein & Bloom, 1977). The requirement for complement in antibody-mediated demyelination in vivo can be studied in animals depleted of components C3 and C5 by treatment with cobra venom factor (CVF). CVF binds factor B and forms a stable C3/C5 convertase, resulting in the rapid consumption of these components and depletion of serum haemolytic complement activity. CVF has previously been used to examine the role of complement in the pathogenesis of EAE and ADEAE (Levine, Cochrane &
INTRODUCTION Experimental allergic encephalomyelitis (EAE) is an acute T cell-mediated autoimmune disease of the CNS (Ortiz-Ortiz & Weigle, 1976), which in the Lewis rat is characterized by perivascular and meningeal inflammation associated with bloodbrain barrier dysfunction and focal but limited demyelination (Lassmann et al., 1988). However, demyelination can be greatly augmented by intravenous injection of the CNS myelin-specific monoclonal antibody (MoAb) 8-18C5 (Linington, Webb & Woodhams, 1984; Schluesner et al., 1987; Linington et al., 1988). In this model of acute antibody-mediated demyelinating EAE (ADEAE), the MoAb enters the CNS when blood-brain barrier function is compromised and binds to an epitope of the myelin oligodendrocyte glycoprotein (MOG) exposed on the outer surface of the myelin sheath (Linington et al., 1988; Brunner et al., 1989). However, the mechanism by which this Correspondence: Sara Piddlesden, Section of Neurology, Department of Medicine, University of Wales College of Medicine, Heath
Park, Cardiff CF4 4XN, UK.
245
S. Piddlesden et al.
246
Carpenter, 1971; Pabst et al., 1971; Morariau & Dalmasso, 1978; Linington et al., 1989b). In these later studies, depletion of serum complement with CVF led to the suppression of lowgrade clinical disease without substantially reducing CNS inflammation, and a reduced ability of the MoAb 8-18C5 to initiate demyelination in ADEAE. However, these studies were essentially qualitative and the animals were killed and examined histologically at a relatively late stage, at a time when serum C3/C5 levels would have been recovered following the clearance of CVF from the circulation (Linington et al., 1989b). In view of the known potency of complement-derived factors such as C3a and C5a as pro-inflammatory and chemotactic agents (Hollerhage, Walter & Stolke, 1989), the presence of CNS inflammation or demyelination 3 days or later after CVF treatment may reflect a delay in the recruitment of inflammatory cells, whose subsequent appearance mirrors the rise in serum C3/C5 levels. In view of these uncertainties, we have re-examined the role of terminal complement components in the pathogenesis of demyelination in ADEAE. Two injections of CVF separated by 72 h were used to maintain depletion of serum C3/C5 during the effector stage of actively induced disease, 9-12 days after immunization with myelin basic protein (MBP). The effects of CVF treatment were then quantified with respect to inflammation, C9 deposition, demyelination and the composition of the cellular infiltrates. This quantitative study demonstrates that although complement activation within the CNS plays an essential role in disease following a minimal encephalitogenic challenge, antibody-mediated demyelination in ADEAE can occur independently of C3/C5 activation and MAC formation. MATERIALS AND METHODS Animals and reagents Lewis rats weighing 150-180 g were obtained from Harlan Olac (Hull, UK). MBP was purified from whole guinea-pig brain (Eylar & Jackson, 1974) and stored lyophilized at -20'C. CVF was isolated from lyophilized venom (Naja naja kaouthia; Sigma Chemical Co., Poole, UK) by the method of Vogel & Mueller-Eberhard (1984) and was functionally active both in vivo and in vitro. Polyclonal mouse IgG (pIgG) and the MOGspecific MoAb 8-18C5 (Linington et al., 1984) were purified by sodium sulphate precipitation from pooled normal mouse serum and mouse ascitic fluid, respectively, and stored at approximately 5 mg/ml in phosphate-buffered saline (PBS) at -20°C. The purity of these IgG preparations was monitored by SDS-PAGE under reducing and non-reducing conditions. Induction of disease Lewis rats were injected subcutaneously in the dorsal foot-pads with MBP in 50 ,l of PBS emulsified with an equal volume of Freund's complete adjuvant (FCA; Difco) containing Mycobacterium tuberculosis H37RA (Difco) to induce acute inflammatory EAE. Initial experiments were performed to re-establish the most appropriate immunization protocol to study the clinical suppression of acti-vely induced EAE by CVF. Lewis rats were immunized with either 100 pg MBP in normal FCA, 50 pg MBP in FCA containing extra 200 pg of M. tuberculosis H37RA, or 50 pg MBP in FCA containing extra 100 pg of M. tuberculosis. The protocol chosen for the decomplementation study was that of immunization with 50 pg of MBP in 50 pl PBS,
emulsified with 50 p1 FCA containing 2 mg/ml M. tuberculosis H37RA. The acute demyelinating form of this disease (ADEAE) was induced by i.v. injection of 5 mg MoAb 8-18C5 in I ml of PBS 10 days after sensitization with MBP. EAE controls received an equivalent dose of pIgG. Groups of rats with either EAE or ADEAE were decomplemented by injection with CVF (I pg/g body weight, intravenously) given 9 and 12 days after immunization with MBP. Serum complement levels were monitored using a standard haemolysis assay. Controls were injected with an equal volume of Tris/saline. Rats were weighed and monitored daily for clinical signs of disease which were scored as follows: 1, tail atony; 2, hind-limb weakness; 3, hind-limb paralysis; 4, moribund; and 5, dead. Histopathology and immunostaining Animals were killed 13 days post-immunization by perfusion via the aorta with cold 4% paraformaldehyde in PBS under terminal anaesthesia. The brains and spinal cords were dissected, post-fixed in 4% paraformaldehyde in PBS at 4°C overnight and the tissue then embedded in paraffin. Quantitative analyses were performed on multiple sections of lumbar and cervical spinal cord sections. Inflammation and demyelination were assessed on sections stained with haematoxylin and eosin and Kluver's myelin stain, respectively. The inflammatory incidence was calculated as the number of perivascular infiltrates per spinal cord cross-section, and demyelination was assessed in longitudinal sections of the most severely affected area, the sub-pial region of lumbar cord, by counting the number of points of a 100-point ocular mesh overlying demyelinated areas using a x 100 objective. In each case at least 20 high-power fields were examined. MAC deposition within the CNS was detected immunohistochemically using a rabbit anti-rat C9 antiserum (Linington et al., 1989a) and expressed as the number of peri-vascular deposits of granular C9 immunoreactive material per spinal cord cross-section. Similarly, C3 and C8 deposition were also examined using polyclonal rabbit anti-rat C3 and anti-rat C8 (Jones, Laffafian & Morgan, 1990) antisera. Infiltrating inflammatory cells were identified and quantified using a panel of MoAbs in conjunction with the immunoperoxidase method on sections counterstained with haematoxylin as described previously (Lassmann et al., 1988). In each case cells were counted in 20 objective fields ( x 100) of lumbar and of cervical spinal cord transverse sections. The MoAbs used in this study were: ED 1, which recognizes the majority of macrophage sub-populations in the rat (Dijkstra et al., 1985); ED2, which recognizes a subpopulation of rat macrophages (Dijkstra et al., 1985); and W3/13, which recognizes rat T cells and granulocytes (Williams, Galfre & Milstein, 1977), W3/13 + granulocytes being differentiated from the W3/13+ T cell population by their nuclear morphology.
Statistical analysis The statistical significance of the differences observed between CVF-treated and normocomplementaemic groups of rats with respect to cell counts, complement deposition, demyelination and inflammation was assessed within the framework of the
Kruskal-Wallis test by non-parametric multiple comparisons.
Antibody-mediated demyelination in EAE
a-^:'*.,t:myRe9*B|l!i;:>..}',:
Clin. exp. Immunol. (1991) 83, 245-250
Antibody-mediated demyelination in experimental allergic encephalomyelitis is independent of complement membrane attack complex formation S. PIDDLESDEN, H. LASSMANNt, I. LAFFAFIAN*, B. P. MORGAN* & C. LININGTON: Section of Neurology, Department of Medicine, and *Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, Wales, tInstitute of Brain Research, Austrian Academy of Science, Vienna, Austria, and tDepartment of Neuroimmunology, Max-Planck Institute for Psychiatry, Munich, Germany
(Acceptedfor publication 21 September 1990)
SUMMARY The effects of decomplementation by cobra venom factor (CVF) on the pathogenesis of inflammation and demyelination in experimental allergic encephalomyelitis (EAE) and acute antibody-mediated demyelinating EAE (ADEAE) have been quantified histologically and immunocytochemically. In rats immunized with 50 yg of myelin basic protein in Freund's complete adjuvant containing 100 yug heat-killed Mycobacterium tuberculosis H37Ra, clinical signs of EAE were completely suppressed by two injections of CVF given 9 and 12 days post-immunization. Suppression of clinical disease was associated with a dramatic reduction in peri-vascular inflammation in the CNS, although immunohistochemical staining identified small numbers of infiltrating T cells and macrophages. In contrast, CVF treatment had no significant effect on the clinical severity of ADEAE and although C9 deposition within the CNS was virtually abolished, there was no statistically significant decrease in the extent of demyelination or inflammation. These observations indicate that in the absence of complement components C3 and C5 an antibody-dependent cell-mediated cytotoxic response plays an important role in the pathogenesis of antibody-mediated demyelination. The major role of the complement cascade in EAE appears to be the generation of pro-inflammatory factors that enhance the inflammatory response within the CNS in animals facing a mild encephalitogenic challenge.
Keywords
demyelination complement inflammation experimental allergic encephalomyelitis
anti-myelin antibody subsequently initiates demyelination is unclear. MoAb 8-18C5 is of the IgGI subclass and is capable of complement fixation (Linington et al., 1989b). Complementdependent mechanisms involving complement activation by the 8-18C5 MoAb and membrane attack complex (MAC) formation have been shown to be cytolytic to MOG+ oligodendrocytes in vitro (Linington et al., 1 989b), and the antibody initiates extensive C9 deposition in the white matter tracts of animals with ADEAE (Linington et al., 1989a). However, other studies have indicated that antibody-mediated demyelination in vivo involves an antibody-dependent cellular cytotoxicity (ADCC) mechanism (Brosnan, Bornstein & Bloom, 1977). The requirement for complement in antibody-mediated demyelination in vivo can be studied in animals depleted of components C3 and C5 by treatment with cobra venom factor (CVF). CVF binds factor B and forms a stable C3/C5 convertase, resulting in the rapid consumption of these components and depletion of serum haemolytic complement activity. CVF has previously been used to examine the role of complement in the pathogenesis of EAE and ADEAE (Levine, Cochrane &
INTRODUCTION Experimental allergic encephalomyelitis (EAE) is an acute T cell-mediated autoimmune disease of the CNS (Ortiz-Ortiz & Weigle, 1976), which in the Lewis rat is characterized by perivascular and meningeal inflammation associated with bloodbrain barrier dysfunction and focal but limited demyelination (Lassmann et al., 1988). However, demyelination can be greatly augmented by intravenous injection of the CNS myelin-specific monoclonal antibody (MoAb) 8-18C5 (Linington, Webb & Woodhams, 1984; Schluesner et al., 1987; Linington et al., 1988). In this model of acute antibody-mediated demyelinating EAE (ADEAE), the MoAb enters the CNS when blood-brain barrier function is compromised and binds to an epitope of the myelin oligodendrocyte glycoprotein (MOG) exposed on the outer surface of the myelin sheath (Linington et al., 1988; Brunner et al., 1989). However, the mechanism by which this Correspondence: Sara Piddlesden, Section of Neurology, Department of Medicine, University of Wales College of Medicine, Heath
Park, Cardiff CF4 4XN, UK.
245
S. Piddlesden et al.
246
Carpenter, 1971; Pabst et al., 1971; Morariau & Dalmasso, 1978; Linington et al., 1989b). In these later studies, depletion of serum complement with CVF led to the suppression of lowgrade clinical disease without substantially reducing CNS inflammation, and a reduced ability of the MoAb 8-18C5 to initiate demyelination in ADEAE. However, these studies were essentially qualitative and the animals were killed and examined histologically at a relatively late stage, at a time when serum C3/C5 levels would have been recovered following the clearance of CVF from the circulation (Linington et al., 1989b). In view of the known potency of complement-derived factors such as C3a and C5a as pro-inflammatory and chemotactic agents (Hollerhage, Walter & Stolke, 1989), the presence of CNS inflammation or demyelination 3 days or later after CVF treatment may reflect a delay in the recruitment of inflammatory cells, whose subsequent appearance mirrors the rise in serum C3/C5 levels. In view of these uncertainties, we have re-examined the role of terminal complement components in the pathogenesis of demyelination in ADEAE. Two injections of CVF separated by 72 h were used to maintain depletion of serum C3/C5 during the effector stage of actively induced disease, 9-12 days after immunization with myelin basic protein (MBP). The effects of CVF treatment were then quantified with respect to inflammation, C9 deposition, demyelination and the composition of the cellular infiltrates. This quantitative study demonstrates that although complement activation within the CNS plays an essential role in disease following a minimal encephalitogenic challenge, antibody-mediated demyelination in ADEAE can occur independently of C3/C5 activation and MAC formation. MATERIALS AND METHODS Animals and reagents Lewis rats weighing 150-180 g were obtained from Harlan Olac (Hull, UK). MBP was purified from whole guinea-pig brain (Eylar & Jackson, 1974) and stored lyophilized at -20'C. CVF was isolated from lyophilized venom (Naja naja kaouthia; Sigma Chemical Co., Poole, UK) by the method of Vogel & Mueller-Eberhard (1984) and was functionally active both in vivo and in vitro. Polyclonal mouse IgG (pIgG) and the MOGspecific MoAb 8-18C5 (Linington et al., 1984) were purified by sodium sulphate precipitation from pooled normal mouse serum and mouse ascitic fluid, respectively, and stored at approximately 5 mg/ml in phosphate-buffered saline (PBS) at -20°C. The purity of these IgG preparations was monitored by SDS-PAGE under reducing and non-reducing conditions. Induction of disease Lewis rats were injected subcutaneously in the dorsal foot-pads with MBP in 50 ,l of PBS emulsified with an equal volume of Freund's complete adjuvant (FCA; Difco) containing Mycobacterium tuberculosis H37RA (Difco) to induce acute inflammatory EAE. Initial experiments were performed to re-establish the most appropriate immunization protocol to study the clinical suppression of acti-vely induced EAE by CVF. Lewis rats were immunized with either 100 pg MBP in normal FCA, 50 pg MBP in FCA containing extra 200 pg of M. tuberculosis H37RA, or 50 pg MBP in FCA containing extra 100 pg of M. tuberculosis. The protocol chosen for the decomplementation study was that of immunization with 50 pg of MBP in 50 pl PBS,
emulsified with 50 p1 FCA containing 2 mg/ml M. tuberculosis H37RA. The acute demyelinating form of this disease (ADEAE) was induced by i.v. injection of 5 mg MoAb 8-18C5 in I ml of PBS 10 days after sensitization with MBP. EAE controls received an equivalent dose of pIgG. Groups of rats with either EAE or ADEAE were decomplemented by injection with CVF (I pg/g body weight, intravenously) given 9 and 12 days after immunization with MBP. Serum complement levels were monitored using a standard haemolysis assay. Controls were injected with an equal volume of Tris/saline. Rats were weighed and monitored daily for clinical signs of disease which were scored as follows: 1, tail atony; 2, hind-limb weakness; 3, hind-limb paralysis; 4, moribund; and 5, dead. Histopathology and immunostaining Animals were killed 13 days post-immunization by perfusion via the aorta with cold 4% paraformaldehyde in PBS under terminal anaesthesia. The brains and spinal cords were dissected, post-fixed in 4% paraformaldehyde in PBS at 4°C overnight and the tissue then embedded in paraffin. Quantitative analyses were performed on multiple sections of lumbar and cervical spinal cord sections. Inflammation and demyelination were assessed on sections stained with haematoxylin and eosin and Kluver's myelin stain, respectively. The inflammatory incidence was calculated as the number of perivascular infiltrates per spinal cord cross-section, and demyelination was assessed in longitudinal sections of the most severely affected area, the sub-pial region of lumbar cord, by counting the number of points of a 100-point ocular mesh overlying demyelinated areas using a x 100 objective. In each case at least 20 high-power fields were examined. MAC deposition within the CNS was detected immunohistochemically using a rabbit anti-rat C9 antiserum (Linington et al., 1989a) and expressed as the number of peri-vascular deposits of granular C9 immunoreactive material per spinal cord cross-section. Similarly, C3 and C8 deposition were also examined using polyclonal rabbit anti-rat C3 and anti-rat C8 (Jones, Laffafian & Morgan, 1990) antisera. Infiltrating inflammatory cells were identified and quantified using a panel of MoAbs in conjunction with the immunoperoxidase method on sections counterstained with haematoxylin as described previously (Lassmann et al., 1988). In each case cells were counted in 20 objective fields ( x 100) of lumbar and of cervical spinal cord transverse sections. The MoAbs used in this study were: ED 1, which recognizes the majority of macrophage sub-populations in the rat (Dijkstra et al., 1985); ED2, which recognizes a subpopulation of rat macrophages (Dijkstra et al., 1985); and W3/13, which recognizes rat T cells and granulocytes (Williams, Galfre & Milstein, 1977), W3/13 + granulocytes being differentiated from the W3/13+ T cell population by their nuclear morphology.
Statistical analysis The statistical significance of the differences observed between CVF-treated and normocomplementaemic groups of rats with respect to cell counts, complement deposition, demyelination and inflammation was assessed within the framework of the
Kruskal-Wallis test by non-parametric multiple comparisons.
Antibody-mediated demyelination in EAE
a-^:'*.,t:myRe9*B|l!i;:>..}',:
