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Complement depletion suppresses Lewis rat experimental allergic neuritis
Brain Research, 419 (1987) 97-103 Elsevier
97
BRE 12801
Complement depletion suppresses Lewis rat experimental allergic neuritis* T.E. Feasby 2, J.J. Gilbert 1'3, A.F. Hahn 3 and M. Neilson 2 1Departmentof ClinicalNeurologicalSciences and Department of Pathology, 2UniversityHospitaland 3VictoriaHospital, University of Western Ontario, London, Ont., (Canada) (Accepted 20 January 1987)
Key words: Experimental allergic neuritis; Complement; Demyelination
Lewis rats immunized with myelin and complete Freund's adjuvant were treated with cobra venom factor (CVF) which depletes the C3 component of complement. CVF given at day 9 delayed the onset of experimental allergic neuritis (EAN) by 2-3 days and when given at days 9 and 12 delayed the onset of EAN by 4-5 days. Lumbar nerve roots of CVF-treated rats had significantlyless demyelination than those from control EAN rats. INTRODUCTION The immunological mechanisms of demyelination in experimental allergic neuritis (EAN) remain controversial, some evidence favouring humoural factors 7'24 and some favouring cellular immunity 1'a1'1739. The complement system might be involved in antibody-mediated demyelination 7,24 but recent studies suggest that complement itself might independently initiate or facilitate demyelination 23n,29. The present study examined the effect of depletion of complement on the development of EAN in the Lewis rat. Complement depletion delayed development of the disease and reduced the amount of demyelination. MATERIALS AND METHODS Myelin was isolated from flesh bovine nerve roots using the method of Norton and Poduslo 22 as modified by Kadlubowski et al. 12. Male Lewis rats (175-200 g) were each injected in the foot pads with 5 mg of bovine root myelin plus complete Freund's adjuvant. The rats were weighed daily and graded on
a neurological scale from 0 (normal) to 6 (dead) 28 until sacrifice at day 20. Two groups (A and B) of 10 rats each were treated with cobra venom factor (CVF) (Cordis Laboratories, Miami, FL) 250 U/kg, i.p. Rats in group A were injected with CVF on day 9 postimmunization and rats in group B were injected on days 9 and 12. One control group (C; 6 rats) was not immunized or treated with CVF and a second control group (D; 6 rats) was immunized with myelin and CFA and was given 1 ml 0.9% NaCI instead of CVF. Three separate groups of 5 rats each, corresponding to groups A, B and D were used to assess the effect of CVF on complement activity. Blood (1 ml) was drawn by cardiac puncture on days 10, 13, 16, 18 and 20. Total hemolytic complement activity was assessed using standard methods 2°. Ten additional rats were divided into an untreated EAN group (like group D) and an E A N group treated with CVF at days 9 and 12 (like group B). These rats were sacrificed at day 14 by perfusion with glutaraldehyde. Lumbosacral nerve roots were embedded in Epon and semi-thin sections were stained with Toluidine blue. Five cross-sections of roots from
* A preliminary report has been published (ref. 3). Correspondence: T.E. Feasby, University Hospital, Box 5339, Stn. A., London, Ont., N6A 5A5, Canada. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
98 THE
EFFECT
OF
COBRA
VENOM
FACTOR
ON
EAN
IN
THE
LEWIS
RAT
4-
C
T
i/ I I L\!~! T/i I T T;T,~!
;; ;:':~
3-
L I N I
C A t
"~
2-
G R A D E
/
T/I Ix T./ --" /] //; ./_.i J ~ Il; 'I.'-z/ 020 DAYS
POST-INOCULATION
Fig. 1. Clinical grades of EAN rats.
THE EFFECT OF COBRA VENOM
FACTOR ON EAN IN THE
LEWIS
110-
T
T T r / ~
/
~'L
RAT
x/.
n'/'~ x
L
/.-" XT"""" J.
x ~ . .
~"
-.-
__~..,---!
J.
T
100%OF
"
DAY9 WEIGHT
904
Normal C o n t r o l
s
~v,
",-%
l
, - - - - - . ~,
'~
r
J
l~
l~I
T
o
I
• CVF Da, ,,12
l~------..
;
l~T/i J.
80-
DAYS
I,
I
_
-'~
I
l ~ l l ~
I~T.__T
no
Fig, 2. Weights as a % of day-9 weight.
T
POST-INOCULATION
Ti
.~.---
i
i
99 proached that in the untreated control EAN group at days 19-20. Weight loss in both groups treated with CVF began about the same time as in the untreated EAN control group D (Fig. 2). It never became as severe as in group D. Total hemolytic complement was depressed to about 20% of normal at days 10 and 13 in both groups of animals injected with CVF at day 9 (Fig. 3). By day 16, the complement activity was normal in rats given CVF at day 9 and had risen to 70% of normal in rats given CVF at days 9 and 12. Complement levels were normal in all groups by day 18. Multiple sections of lumbosacral nerve roots showed evidence of inflammatory infiltration and axonal degeneration in both untreated EAN rats and those given CVF at days 9 and 12 (Fig. 4, Table I). However, although demyelination was prominent in the nerve roots of untreated EAN rats, it was virtual-
each rat were read 'blindly' and graded for inflammatory infiltration, axonal degeneration and demyelination on a 0-4 scale. RESULTS Control EAN animals (group D) began to develop clinical signs (limp tail and paraparesis) on day 12 postimmunization (Fig. 1) and began to lose weight at days 11-12 (Fig. 2, weights expressed as % of day 9 weight). The clinical disease reached a peak about day 16. Apparent improvement on days 19 and 20 was spurious as two animals in this group died on day 18 and were not subsequently included in the graph. Animals treated with CVF had delayed onset of symptoms and less severe disease (Fig. 1). The disease began at day 14 in rats given CVF on day 9 and on days 15-16 in rats given CVF on days 9 and 12. Disease severity in the two CVF-treated groups apCOMPLEMENT
LEVELS
T
CHso% 100-
i
80-
.! /I
\=
1 60-
O
Normal
Control
•
Saline
Days 9 &12
II
CVF
Day 9
A
CVF
D a y s 9 & 12
40-
20-
IIA I 12
I 14
DAYS
Fig. 3. Total hemolyticcomplementlevels.
POST-INOCULATION
I 16
118
2~
)J ~ !!
ii!i
ilii
i:i~i
'
101 TABLE I
Inflammation, axonal degeneration and demyelination The symptoms are graded on a 0 - 4 scale on 5 cross-sections of lumbosacral nerve roots from each animal, read 'blindly'. Clinical grades are rated on a 0 - 6 scale 2s.
Rats
CVF group 1 2 3 4 5 Control group 1 2 3 4 5
Clinical grade
lnflammation
Axonal Demyelidegener- nation ation
3 0 2 0 0
3 2 3 2 2
3 1 3 1 0
0 0 0 0 1
4 4 4 5 5
1 2 2 2 1
2 3 2 4 3
0 2 1 2 2
ly absent in those treated with CVF at days 9 and 12 (P < 0.01). DISCUSSION
CVF which depletes the C 3 component of complement, thereby interfering with the cascade of complement activation, reduced the severity of Lewis rat EAN. The delay in onset of disease corresponded well with the reduction in total hemolytic complement activity in the blood. The degree of inflammatory infiltration in the nerve roots of these rats was not affected by CVF but the demyelination was inhibited almost completely. Experimental allergic encephalomyelitis (EAE) in the guinea pig has also been inhibited with CVF 21,23. Injection of CVF at days 0 and 4 postimmunization delayed the onset of EAE by about 5 days and reduced its severity for up to 50 days2x. Conversely, it was found that EAE developed in a normal fashion after myelin and CFA inoculation in guinea pigs with congenital absence of C 4. Levine et al. TM found that CVF has only minor effects on the hyperacute form
of Lewis rat EAE. Gerwin and Frank 5 found that EAN developed normally in congenitally C4 deficient guinea pigs. Two guinea pigs who were depleted of C3 by using CVF-developed ventral root inflammatory lesions. No details of clinical disease were reported. The mechanism by which CVF inhibits the development of Lewis rat EAN is uncertain but our pathological studies reveal that demyelination is reduced in the nerve roots at a time when the disease is delayed. C 3 depletion could delay demyelination by blocking antigen-antibody complex-induced activation of complement. This has been demonstrated in rabbit EAN serum-induced demyelination7'24 where treatment of recipient rats with CVF and heating rabbit EAN serum to 56 °C for 30 min prevented demyelination in the intraneural injection model. Demyelination in the rabbit model of EAN is probably mediated by antigalactocerebroside antibodies 25. In the Lewis rat, however, while there is strong evidence for cell-mediated demyelination 11'19, the evidence for antibody-mediated demyelination is scanty. Injection of Lewis rat EAN serum into Wistar rat sciatic nerves has not produced significant demyelination (Feasby et al., unpublished). Antibodies to P2protein, a component of peripheral nerve myelin (PNM), have been detected in myelin-induced Lewis rat EAN, but a significant rise in titre did not occur until after the onset of clinical disease 11. Circulating immune complexes have been detected in Lewis ratEAN but only two weeks postimmunization, after the onset of clinical disease 13. The lack of complement might directly inhibit demyelination. Myelin has recently been shown to activate complement in the absence of antibody. Vanguri et al. 29 showed that isolated rat and human central nervous system myelin activates complement by the classical pathway and does not activate the alternative pathway. Conversely, Koski et al. 14 showed that human PNM activates the alternative but not the classical complement pathway. They found that the Po PNM protein could activate the alternative pathway when it was inserted into an artificial lipid mem-
Fig. 4. a: representative l u m b a r nerve root from E A N rat treated with saline at days 9 and 12, showing evidence of inflammation, axonal damage and demyelination (arrows). x765. b: representative lumbar nerve root from E A N rat treated with C V F at days 9 and 12. Note similar changes but little demyelination, x765.
102 brane suggesting that it might be the complement-activating factor in PNM. Possibly, the depletion of C 3 blocks activation of complement that exposed myelin might ordinarily initiate by the alternative pathway 14. Myelin would be exposed to complement components early in the course of E A N with breakdown of the b l o o d - n e r v e barrier 8'14. However, it should be noted that in previous work by ourselves 4 and others 24 intraneural injection into Wistar rat sciatic nerve of normal rat serum plus guinea pig serum (both presumably containing complement) did not result in significant demyelination. Complement may also participate in demyelination by potentiating the demyelinating effect of enzymes such as plasmin and phospholipases, some of which are secreted by activated macrophages 2. Serum deficient in complement components C 3 or C4 did not potentiate myelin breakdown in vitro 2. Thus C 3 depletion by C V F could indirectly inhibit demyelination.
REFERENCES 1 Astrom, K., Webster, H. and Arnason, B., The initial lesion in experimental allergic neuritis: a phase and electron microscopic study, J. Exp. Med., 128 (1968) 469-482. 2 Cammer, W., Brosnan, C., Basile, C., Bloom, B. and Norton, W., Complement potentiates the degradation of myelin proteins by plasmin: implications for a mechanism of inflammatory demyelination, Brain Research, 364 (1986) 91-101, 3 Feasby, T., Gilbert, J., Hahn, A. and Neilson, M., Complement depletion suppresses Lewis rat EAN, Can. J. Neurol. Sci., 12 (1985) 205. 4 Feasby, T., Hahn, A. and Gilbert, J., Passive transfer studies in Guillain-Barre polyneuropathy, Neurology, 32 (1982) 1159-1167. 5 Gerwin, R, and Frank, M., Complement independence of experimental allergic neuritis (EAN), J. Neuropathol. Exp. Neurol., 34 (1975) 98. 6 Guillain-Barre Syndrome Study Group, Plasmapheresis and acute GuiUain-Barre syndrome, Neurology, 35 (1985) 1096-1104. 7 Hahn, A., Gilbert, J. and Feasby, T., Passive transfer of demyelination by experimental allergic neuritis serum, Acta Neuropathol., 49 (1980) 169-176. 8 Hahn, A., Feasby, T. and Gilbert, J., Blood-nerve barrier studies in experimental allergic neuritis, Acta Neuropathol., 68 (1985) 101-109. 9 Harrison, B., Hansen, L., Pollard, J. and McLeod, J., Demyelination induced by serum from patients with GuillainBarre syndrome, Ann. Neurol., 15 (1984) 163-170. 10 Hartung, H.P., Schwenke, C. and Toyka, K., Elevated levels of complement activation products C3a and Csa in CSF of patients with the GuiUain-Barre syndrome, Neurology, 36, Suppl. 1 (1986) 186.
These observations may have some relevance for human disease. Although the mechanisms are as yet unknown, antibody-mediated demyelination has been implicated in the pathogenesis of Guillain-Barre polyneuropathy (GBP) 4,6'9,15'26'27, the human analogue of E A N . Recently, evidence of complement activation has been found in the serum 16 and cerebrospinal fluid 1° of patients with GBP. These observations plus our findings in E A N suggest that complement may play a role in both experimental and human demyelination. ACKNOWLEDGEMENTS We thank Elizabeth Leslie, David Lovgren and A n n Calarco for excellent assistance and Dr. W. Chodirker for helping with the complement assays. Supported by the Muscular Dystrophy Association of Canada and the Medical Research Council of Canada.
11 Hughes, R., Kadlubowski, M., Gray, 1. and Leibowitz, S.. Immune responses in experimental allergic neuritis, J. Neurol. Neurosurg. Psychiatr., 44 (1981) 565-569. 12 Kadlubkowski, M., Hughes, R. and Gregson, N., Experimental allergic neuritis in the Lewis rat - - characterization of the activity of peripheral myelin and its major basic protein P2, Brain Research, 184 (1980)439-454. 13 Koh, C.S., Nakano, T., Tsukada, N., Yanagisawa, N., Okano, A. and Taketomi, T., Detection of immune complexes in experimental allergic neuritis, J. Neurol. Sci., 63 (1984) 229-239. 14 Koski, C., Vanguri, P. and Shin, M., Activation of the alternative pathway of complement by human peripheral nerve myelin, J. Immunol., 134 (1985) 1810-1814. 15 Koski, C., Humphrey, R. and Shin, M., Anti-peripheral myelin antibody in patients with demyelinating neuropathy: quantitative and kinetic determination of serum antibody by complement component 1 fixation, Proc. Natl. Acad. Sci. U.S.A.. 82 (1985) 905-909. 16 Koski, C.. Sanders, M., Swoveland, P., Shin, M., Frank. M. and Joiner, K., Quantitation of C9 neoantigen in the serum of patients with Gnillain-Barre syndrome and other demyelinating neuropathies. Neurology, 36 (Suppl. 1) (1986) 304. 17 Lampert, P., Mechanism of demyelination in experimental allergic neuritis. Electron microscopic studies, Lab. Invest., 20 (1969) 127-138. 18 Levine, S., Cochrane, C., Carpenter, C. and Behan, P., Allergic encephalomyelitis: effect of complement depletion with cobra venom (35880), Proc. Soc. Exp. BioL Med.. 138 (1971) 285-289. 19 Lirmington, C., Izumo, S., Suzuki, M., Uyemura, K., Meyermann, R. and Wekerle, H., A permanent rat T cell line that mediates experimental allergic neuritis in the Lewis rat in vivo, J. lmmunol., 133 (1984) 1946-1950.
103 20 Kabat, E.A. and Mayer, M.M., Experimental Immunochemistry, 2nd edn., Thomas, Springfield, IL 1971, pp. 133-240. 21 Morariu, M. and Dalmasso, A., Experimental allergic encephalomyelitis in cobra venom factor-treated and C4-deficient guinea pigs, Ann. Neurol., 4 (1978) 427-430. 22 Norton, W. and Poduslo, S., Myelination in rat brain - changes in myelin composition during brain maturation, J. Neurochem., 21 (1973) 759-773. 23 Pabst, H., Day, N. and Gewurz, H. et al., Prevention of experimental allergic encephalomyelitis with cobra venom factor, Proc. Soc. Exp. Biol. Med., 136 (1971) 555-560. 24 Saida, T., Saida, K., Silberberg, D., Brown, M., Transfer of demyelination by intraneural injection of experimental allergic neuritis serum, Nature (London), 272 (1978) 639-641. 25 Saida, T., Saida, K., Silberberg, D. and Brown M., Experi-
26
27
28
29
mental allergic neuritis induced by galactocerebroside, Ann. Neurol., 9 Suppl., (1981) 87-101. Saida, T., Saida, K., Lisak, R., Brown, M., Silberberg, D. and Asbury, A., In vivo demyelinating activity of sera from patients with Guillain-Barre syndrome, Ann. Neurol., 11 (1982) 69-75. Sumner, A., Said, G., Idy, I. and Metral, S., Syndrome de Guillain-Barre: effets electrophysiologiques et morphologiques du serum humain introduit dans l'espace endoneural du nerf sciatique du rat, Rev. Neurol., 138 (1982) 17-24. Tansey, F. and Brosnan, C., Protection against experimental allergic neuritis with silica quartz dust, J. Neuroimmunol., 3 (1982) 168-179. Vanguri, P,, Koski, C., Silverman, B. and Shin, M., Complement activation by isolated myelin: activation of the classical pathway in the absence of myelin-specificantibodies, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 3290-3294.
97
BRE 12801
Complement depletion suppresses Lewis rat experimental allergic neuritis* T.E. Feasby 2, J.J. Gilbert 1'3, A.F. Hahn 3 and M. Neilson 2 1Departmentof ClinicalNeurologicalSciences and Department of Pathology, 2UniversityHospitaland 3VictoriaHospital, University of Western Ontario, London, Ont., (Canada) (Accepted 20 January 1987)
Key words: Experimental allergic neuritis; Complement; Demyelination
Lewis rats immunized with myelin and complete Freund's adjuvant were treated with cobra venom factor (CVF) which depletes the C3 component of complement. CVF given at day 9 delayed the onset of experimental allergic neuritis (EAN) by 2-3 days and when given at days 9 and 12 delayed the onset of EAN by 4-5 days. Lumbar nerve roots of CVF-treated rats had significantlyless demyelination than those from control EAN rats. INTRODUCTION The immunological mechanisms of demyelination in experimental allergic neuritis (EAN) remain controversial, some evidence favouring humoural factors 7'24 and some favouring cellular immunity 1'a1'1739. The complement system might be involved in antibody-mediated demyelination 7,24 but recent studies suggest that complement itself might independently initiate or facilitate demyelination 23n,29. The present study examined the effect of depletion of complement on the development of EAN in the Lewis rat. Complement depletion delayed development of the disease and reduced the amount of demyelination. MATERIALS AND METHODS Myelin was isolated from flesh bovine nerve roots using the method of Norton and Poduslo 22 as modified by Kadlubowski et al. 12. Male Lewis rats (175-200 g) were each injected in the foot pads with 5 mg of bovine root myelin plus complete Freund's adjuvant. The rats were weighed daily and graded on
a neurological scale from 0 (normal) to 6 (dead) 28 until sacrifice at day 20. Two groups (A and B) of 10 rats each were treated with cobra venom factor (CVF) (Cordis Laboratories, Miami, FL) 250 U/kg, i.p. Rats in group A were injected with CVF on day 9 postimmunization and rats in group B were injected on days 9 and 12. One control group (C; 6 rats) was not immunized or treated with CVF and a second control group (D; 6 rats) was immunized with myelin and CFA and was given 1 ml 0.9% NaCI instead of CVF. Three separate groups of 5 rats each, corresponding to groups A, B and D were used to assess the effect of CVF on complement activity. Blood (1 ml) was drawn by cardiac puncture on days 10, 13, 16, 18 and 20. Total hemolytic complement activity was assessed using standard methods 2°. Ten additional rats were divided into an untreated EAN group (like group D) and an E A N group treated with CVF at days 9 and 12 (like group B). These rats were sacrificed at day 14 by perfusion with glutaraldehyde. Lumbosacral nerve roots were embedded in Epon and semi-thin sections were stained with Toluidine blue. Five cross-sections of roots from
* A preliminary report has been published (ref. 3). Correspondence: T.E. Feasby, University Hospital, Box 5339, Stn. A., London, Ont., N6A 5A5, Canada. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
98 THE
EFFECT
OF
COBRA
VENOM
FACTOR
ON
EAN
IN
THE
LEWIS
RAT
4-
C
T
i/ I I L\!~! T/i I T T;T,~!
;; ;:':~
3-
L I N I
C A t
"~
2-
G R A D E
/
T/I Ix T./ --" /] //; ./_.i J ~ Il; 'I.'-z/ 020 DAYS
POST-INOCULATION
Fig. 1. Clinical grades of EAN rats.
THE EFFECT OF COBRA VENOM
FACTOR ON EAN IN THE
LEWIS
110-
T
T T r / ~
/
~'L
RAT
x/.
n'/'~ x
L
/.-" XT"""" J.
x ~ . .
~"
-.-
__~..,---!
J.
T
100%OF
"
DAY9 WEIGHT
904
Normal C o n t r o l
s
~v,
",-%
l
, - - - - - . ~,
'~
r
J
l~
l~I
T
o
I
• CVF Da, ,,12
l~------..
;
l~T/i J.
80-
DAYS
I,
I
_
-'~
I
l ~ l l ~
I~T.__T
no
Fig, 2. Weights as a % of day-9 weight.
T
POST-INOCULATION
Ti
.~.---
i
i
99 proached that in the untreated control EAN group at days 19-20. Weight loss in both groups treated with CVF began about the same time as in the untreated EAN control group D (Fig. 2). It never became as severe as in group D. Total hemolytic complement was depressed to about 20% of normal at days 10 and 13 in both groups of animals injected with CVF at day 9 (Fig. 3). By day 16, the complement activity was normal in rats given CVF at day 9 and had risen to 70% of normal in rats given CVF at days 9 and 12. Complement levels were normal in all groups by day 18. Multiple sections of lumbosacral nerve roots showed evidence of inflammatory infiltration and axonal degeneration in both untreated EAN rats and those given CVF at days 9 and 12 (Fig. 4, Table I). However, although demyelination was prominent in the nerve roots of untreated EAN rats, it was virtual-
each rat were read 'blindly' and graded for inflammatory infiltration, axonal degeneration and demyelination on a 0-4 scale. RESULTS Control EAN animals (group D) began to develop clinical signs (limp tail and paraparesis) on day 12 postimmunization (Fig. 1) and began to lose weight at days 11-12 (Fig. 2, weights expressed as % of day 9 weight). The clinical disease reached a peak about day 16. Apparent improvement on days 19 and 20 was spurious as two animals in this group died on day 18 and were not subsequently included in the graph. Animals treated with CVF had delayed onset of symptoms and less severe disease (Fig. 1). The disease began at day 14 in rats given CVF on day 9 and on days 15-16 in rats given CVF on days 9 and 12. Disease severity in the two CVF-treated groups apCOMPLEMENT
LEVELS
T
CHso% 100-
i
80-
.! /I
\=
1 60-
O
Normal
Control
•
Saline
Days 9 &12
II
CVF
Day 9
A
CVF
D a y s 9 & 12
40-
20-
IIA I 12
I 14
DAYS
Fig. 3. Total hemolyticcomplementlevels.
POST-INOCULATION
I 16
118
2~
)J ~ !!
ii!i
ilii
i:i~i
'
101 TABLE I
Inflammation, axonal degeneration and demyelination The symptoms are graded on a 0 - 4 scale on 5 cross-sections of lumbosacral nerve roots from each animal, read 'blindly'. Clinical grades are rated on a 0 - 6 scale 2s.
Rats
CVF group 1 2 3 4 5 Control group 1 2 3 4 5
Clinical grade
lnflammation
Axonal Demyelidegener- nation ation
3 0 2 0 0
3 2 3 2 2
3 1 3 1 0
0 0 0 0 1
4 4 4 5 5
1 2 2 2 1
2 3 2 4 3
0 2 1 2 2
ly absent in those treated with CVF at days 9 and 12 (P < 0.01). DISCUSSION
CVF which depletes the C 3 component of complement, thereby interfering with the cascade of complement activation, reduced the severity of Lewis rat EAN. The delay in onset of disease corresponded well with the reduction in total hemolytic complement activity in the blood. The degree of inflammatory infiltration in the nerve roots of these rats was not affected by CVF but the demyelination was inhibited almost completely. Experimental allergic encephalomyelitis (EAE) in the guinea pig has also been inhibited with CVF 21,23. Injection of CVF at days 0 and 4 postimmunization delayed the onset of EAE by about 5 days and reduced its severity for up to 50 days2x. Conversely, it was found that EAE developed in a normal fashion after myelin and CFA inoculation in guinea pigs with congenital absence of C 4. Levine et al. TM found that CVF has only minor effects on the hyperacute form
of Lewis rat EAE. Gerwin and Frank 5 found that EAN developed normally in congenitally C4 deficient guinea pigs. Two guinea pigs who were depleted of C3 by using CVF-developed ventral root inflammatory lesions. No details of clinical disease were reported. The mechanism by which CVF inhibits the development of Lewis rat EAN is uncertain but our pathological studies reveal that demyelination is reduced in the nerve roots at a time when the disease is delayed. C 3 depletion could delay demyelination by blocking antigen-antibody complex-induced activation of complement. This has been demonstrated in rabbit EAN serum-induced demyelination7'24 where treatment of recipient rats with CVF and heating rabbit EAN serum to 56 °C for 30 min prevented demyelination in the intraneural injection model. Demyelination in the rabbit model of EAN is probably mediated by antigalactocerebroside antibodies 25. In the Lewis rat, however, while there is strong evidence for cell-mediated demyelination 11'19, the evidence for antibody-mediated demyelination is scanty. Injection of Lewis rat EAN serum into Wistar rat sciatic nerves has not produced significant demyelination (Feasby et al., unpublished). Antibodies to P2protein, a component of peripheral nerve myelin (PNM), have been detected in myelin-induced Lewis rat EAN, but a significant rise in titre did not occur until after the onset of clinical disease 11. Circulating immune complexes have been detected in Lewis ratEAN but only two weeks postimmunization, after the onset of clinical disease 13. The lack of complement might directly inhibit demyelination. Myelin has recently been shown to activate complement in the absence of antibody. Vanguri et al. 29 showed that isolated rat and human central nervous system myelin activates complement by the classical pathway and does not activate the alternative pathway. Conversely, Koski et al. 14 showed that human PNM activates the alternative but not the classical complement pathway. They found that the Po PNM protein could activate the alternative pathway when it was inserted into an artificial lipid mem-
Fig. 4. a: representative l u m b a r nerve root from E A N rat treated with saline at days 9 and 12, showing evidence of inflammation, axonal damage and demyelination (arrows). x765. b: representative lumbar nerve root from E A N rat treated with C V F at days 9 and 12. Note similar changes but little demyelination, x765.
102 brane suggesting that it might be the complement-activating factor in PNM. Possibly, the depletion of C 3 blocks activation of complement that exposed myelin might ordinarily initiate by the alternative pathway 14. Myelin would be exposed to complement components early in the course of E A N with breakdown of the b l o o d - n e r v e barrier 8'14. However, it should be noted that in previous work by ourselves 4 and others 24 intraneural injection into Wistar rat sciatic nerve of normal rat serum plus guinea pig serum (both presumably containing complement) did not result in significant demyelination. Complement may also participate in demyelination by potentiating the demyelinating effect of enzymes such as plasmin and phospholipases, some of which are secreted by activated macrophages 2. Serum deficient in complement components C 3 or C4 did not potentiate myelin breakdown in vitro 2. Thus C 3 depletion by C V F could indirectly inhibit demyelination.
REFERENCES 1 Astrom, K., Webster, H. and Arnason, B., The initial lesion in experimental allergic neuritis: a phase and electron microscopic study, J. Exp. Med., 128 (1968) 469-482. 2 Cammer, W., Brosnan, C., Basile, C., Bloom, B. and Norton, W., Complement potentiates the degradation of myelin proteins by plasmin: implications for a mechanism of inflammatory demyelination, Brain Research, 364 (1986) 91-101, 3 Feasby, T., Gilbert, J., Hahn, A. and Neilson, M., Complement depletion suppresses Lewis rat EAN, Can. J. Neurol. Sci., 12 (1985) 205. 4 Feasby, T., Hahn, A. and Gilbert, J., Passive transfer studies in Guillain-Barre polyneuropathy, Neurology, 32 (1982) 1159-1167. 5 Gerwin, R, and Frank, M., Complement independence of experimental allergic neuritis (EAN), J. Neuropathol. Exp. Neurol., 34 (1975) 98. 6 Guillain-Barre Syndrome Study Group, Plasmapheresis and acute GuiUain-Barre syndrome, Neurology, 35 (1985) 1096-1104. 7 Hahn, A., Gilbert, J. and Feasby, T., Passive transfer of demyelination by experimental allergic neuritis serum, Acta Neuropathol., 49 (1980) 169-176. 8 Hahn, A., Feasby, T. and Gilbert, J., Blood-nerve barrier studies in experimental allergic neuritis, Acta Neuropathol., 68 (1985) 101-109. 9 Harrison, B., Hansen, L., Pollard, J. and McLeod, J., Demyelination induced by serum from patients with GuillainBarre syndrome, Ann. Neurol., 15 (1984) 163-170. 10 Hartung, H.P., Schwenke, C. and Toyka, K., Elevated levels of complement activation products C3a and Csa in CSF of patients with the GuiUain-Barre syndrome, Neurology, 36, Suppl. 1 (1986) 186.
These observations may have some relevance for human disease. Although the mechanisms are as yet unknown, antibody-mediated demyelination has been implicated in the pathogenesis of Guillain-Barre polyneuropathy (GBP) 4,6'9,15'26'27, the human analogue of E A N . Recently, evidence of complement activation has been found in the serum 16 and cerebrospinal fluid 1° of patients with GBP. These observations plus our findings in E A N suggest that complement may play a role in both experimental and human demyelination. ACKNOWLEDGEMENTS We thank Elizabeth Leslie, David Lovgren and A n n Calarco for excellent assistance and Dr. W. Chodirker for helping with the complement assays. Supported by the Muscular Dystrophy Association of Canada and the Medical Research Council of Canada.
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