Acta neurol. scandinav. 60, 140-148, 1979 Medical Research Council, Demyelinating Diseases Unit, Newcastle General Hospitcl, Newcastle upon Tyne, England
Studies on the inactivation of encephalitogenic myelin basic protein by serum J. R. MCDERMOTT AND A. B. KEITH
rhis study confirms previous reports that myelin basic protein loses its encephalitogenic activity when incubated in normal serum at 37" C. The mechanisms for this was studied. 1251-labelledhuman myelin basic protein was rapidly degraded by normal guinea pig serum to low molecular weight products as shown by polyacrylamide gel electrophoresis. An intermediate product of molecular weight about 6000 daltons was seen. Plasma had a much lower degradative activity than serum; the half life of myelin basic protein was 3.8 hours in plasma compared with 12 minutes in serum. Serum degraded myelin basic protein was no longer capable of suppressing experimental allergic encephalomyelitis in the guinea pig nor of eliciting delayedtype hypersensitivity in guinea pigs sensitized to myelin basic protein. Key words: Myelin basic protein - experimental allergic encephalomyelitis - guinea pig serum - encephalitogenicity
The basic protein of the myelin sheath (MBP) induces experimental allergic encephalomyelitis (EAE), a model autoimmune disease of the CNS, when injected into most laboratory animals with Freund's complete adjuvant (FCA). Several studies have shown that MBP loses its encephalitogenicity when incubated prior to injection with normal serum from several species (Lamoureux et al. 1970, Gerstl et al. 1972, Bernard & Lamoureux 1975a, Pescovitz et al. 1978). The inactivation was earlier ascribed to the binding of MBP by serum proteins, chiefly the a-globulin fraction (Bernard & Lamoureux 1975b). Recently, however, Pescovitz et al. (1978) showed that normal rat serum enzymatically degraded MBP to non-encephalitogenic fragments and that this activity was absent in plasma. We report here the results of our own studies using lz5I-MBP which confirm that MBP is degraded by guinea pig serum. We show that, in addition to losing its encephalitogenicity, serum-treated MBP also loses its ability to suppress EAE and its ability to elicit delayed-type hypersensitivity in MBPsensitized guinea pigs.
0001-6314/79/090140-09 $0.2.50/0 @ 1979 Munksgaard, Copenhagen
141 MATERIAL AND METHODS The isolation and iodination of human MBP were described previously (McDermott et al. 1977). The specific activity of lz5I-MBP was about 3 pCi/pg; this corresponds to a substitution ratio of one iodine atom per 30 molecules of MBP. Blood was removed from guinea pigs by cardiac puncture. Serum was obtained after clotting for 1 h at 37" C, and plasma was obtained using heparin; both were used fresh. Degradation of ' Y - M B P lZSI-MBP(5 X lo6 cpm aprrox.) was added to human MBP (2mg/ml) in phosphate buffered (pH 7.2) saline solution (PBSA). 25 p l of this solution was incubated at 37" C for various times with 50 p l of fresh serum or plasma. The reaction was stopped with 0.5 ml of phenol/formic acid (14:3, w/v). 25 p l of this solution was applied to a horizontal slab gel (12 % polyacrylamide) equilibrated in phenoVformic acid/water (14:3:3, w/v/v) and subjected to electrophoresis at 300 v for 5 h. Gels were fixed in glacial acetic acid, stained with 0.25 % amido black in 7 % acetic acid and destained electrophoretically. Cytochrome-C (12,000 daltons) was used as a marker. The gel was cut into strips which were sliced into 2 mm sections. The radioactivity was determined in a Packard Autogamma counter. Serum treatment of MBP for injection The method of Bernard & Lamoureux (1975a) was used. Human MBP (5 mg) was dissolved in PBSA ( 5 ml); this solution was added to fresh serum (10 ml) and incubated at 37" C for 20 h in a sterile capped tube. Animals Hartley and Strain 13 guinea pigs from our own colonies were maintained on commercial pelleted diet and water ad libitum, with fresh cabbage given three times weekly. Animals were injected with equal amounts of the appropriate emulsion into the dorsum of both hind feet. They were weighed and examined daily for signs of EAE, which were scored as previously (McDermott & Keith 1979). Animals were killed when moribund by exsanguination under ether anaesthesia. Brains and spinal cord were r e moved into formalin (10 %) and histological examination performed on haematoxylin/ eosin stained paraffin-embedded sections as before (Keith 1979). EAE activity MBP in an emulsion of saline (0.1 ml) and FCA (0.1 ml; containing 0.1 mg H37Ra) was injected into Hartley guinea pigs. The serumlMBP mixture from above (0.15 ml) was emulsified with FCA (0.1 ml, containing 0.1 mg H37Ra) and injected into Hartley guinea pigs. Supression of EAE Strain 13 guinea pigs (250-350 g) were injected with 0.2 ml of an emulsion of guinea pig spinal cord homogenate (1:l w/v in saline) with FCA (containing 10 mg/ml H37Ra). Serum-treated MBP was emulsified in incomplete Freund's adjuvant - IFA (1:l v/v) and 0.3 ml injected subcutaneously over the nuchal region at the onset of the first clinical signs of the disease. 0.3 ml of the emulsion was given for a further 9 days (total of 0.5 mg of MBP). In a second treatment group, MBP (50 pg) in 0.1 ml of saline/IFA was given as above. A third group was left untreated.
142 In a second experiment, Strain 13 guinea pigs were challenged with MBP (50 pg) in 0.2 ml of saline/FCA (10 mg/ml H37Ra) and treated with MBP/serum as in the first experiment. Delayed-type skin reactivity Guinea pigs challenged with MBP (50 pg) in FCA were tested at 10 days post-sensitisation for delayed-type hypersensitivity by intradermal injection of 0.1 ml of three saline test solutions: MBP (100 pg); MBP/serum (20 h incubation; 100 pg equivalent), serum (2:1, d v ) . Spot diameters were measured at 48 h.
RESULTS
Degradation of MBP by serum and plasma Initial experiments with serum showed that at 24 h no 1251-MBPremained on the gel. The time course of the degradation was determined by integrating the 1251-MBPpeak for various reaction times. The result (Figure 1) shows that the degradation of MBP is considerably more rapid in serum than in plasma (half life of I2T-MBP: 12 min and 3.8 h, respectively). A second 1251-labelledband was present on the gel in the serum-degraded MBP at '/2 h reaction time (Figure 2a). At 20 h this peak was absent and probably represents an intermediate breakdown product of 1251-MBP.The plasma-degraded 1251-MBPdid not have a prominent peak at this position at any of the reaction times (V2, 4, 8 and 20 h) (Figure 2b). In both the serum and plasma experiments, a small peak of radioactivity was seen at the origin of the gel at all time periods. Loss of encephalitogenic activity None of the animals sensitized with serum-treated MBP in CFA developed clinical or histological signs of EAE (Table 1). Compare this with the high encephalitogenicity of untreated human MBP. Suppression of EAE Untreated Strain 13 guinea pigs had an initial episode of the disease at between 10 and 14 days post-sensitization with guinea pig spinal cord/FCA emulsion. Four animals died in the acute phase of the disease and three animals deteriorated progressively over several weeks. Nine of the 16 animals recovered, but eight of these later suffered relapses. In contrast, the guinea pigs treated with MBP had a milder initial episode in which there were no fatalities. Although six of the eight animals later suffered relapses, the majority of the animals were well when killed at times of 9-45 weeks after sensitization. One animal in the group died after a third relapse at 24 weeks post-sensitization.
143
. 2
4
20
8 Reaction time (hours)
Figure 1 . Rate o f degradation of lZ51-MBPin guinea pig serum and plasma (0-0).
Gel slice number
(0-0)
Gel slice number
Figure 2 . Distribution o f radioactivity ( I z s l ) in polyacrylamide gels after incubation of 1251-MBPwith (a) serum and ( b ) plasma. control; - - - - %-h reaction in serum, 4-h reaction in plasma; -._._20-h reaction.
The guinea pigs treated with serum/MBP resembled the untreated controls in the severity of the initial episode of the disease and also in the overall mortality. Four of the six survivors were killed when clinically well at 4-10
144 Table I . Encephalitogenic activity of serum-treated MBP in Harttey guinea pigs
Challenge
No. developing EAE***
MBP (16,ug)* (32 Pug)* MBP (50 pg)/serum**
* ** ***
5/5 5/5 0/6
In an emulsion of Freund's complete adjuvant (containing lmg/ml H37Ra) and saline (1:l). Human MBP held in fresh guinea pig serum for 20 h at 37" C . Clinical and/or histological evidence for EAE. All the positives had severe clinical signs.
months after sensitization. However, all these animals had moderate to severe histological lesions throughout the CNS. Attempted suppression of acute EAE induced by MBP using the serum/ MBP preparation was completely ineffective, four of the five animals following a rapid and progressive course to death. Delayed-type hypersensitivity All nine tested animals had positive skin reactions to MBP; the mean spot diameter was 10 mm. The serum-treated MBP and serum control injections were negative in all cases. DISCUSSION
The finding that MBP loses its encephalitogenic activity when kept in normal serum at 37" C for several hours agrees with previous reports (Lamoureux et al. 1970, Gerstl et al. 1972, Bernard & Lamoureux 1975a, Pescovtitz et al. 1978). In this case, human MBP and guinea pig serum were used, but the factor responsible for the inactivation of MBP is present in sera from other species (Bernard & Lamoureux 1795a). The loss of encephalitogenicity has been attributed to the binding of serum proteins to MBP which inactivated the encephalitogenic site; cu2-macroglobulin, transferin and ceruloplasmin fractions were found to be the most potent inactivators (Bernard & Lamoureux 1975b). These workers found no evidence for degradation of lZ5I-MBPusing Sephadex G25 column chromatography as the means of identifying small molecular weight (
Studies on the inactivation of encephalitogenic myelin basic protein by serum J. R. MCDERMOTT AND A. B. KEITH
rhis study confirms previous reports that myelin basic protein loses its encephalitogenic activity when incubated in normal serum at 37" C. The mechanisms for this was studied. 1251-labelledhuman myelin basic protein was rapidly degraded by normal guinea pig serum to low molecular weight products as shown by polyacrylamide gel electrophoresis. An intermediate product of molecular weight about 6000 daltons was seen. Plasma had a much lower degradative activity than serum; the half life of myelin basic protein was 3.8 hours in plasma compared with 12 minutes in serum. Serum degraded myelin basic protein was no longer capable of suppressing experimental allergic encephalomyelitis in the guinea pig nor of eliciting delayedtype hypersensitivity in guinea pigs sensitized to myelin basic protein. Key words: Myelin basic protein - experimental allergic encephalomyelitis - guinea pig serum - encephalitogenicity
The basic protein of the myelin sheath (MBP) induces experimental allergic encephalomyelitis (EAE), a model autoimmune disease of the CNS, when injected into most laboratory animals with Freund's complete adjuvant (FCA). Several studies have shown that MBP loses its encephalitogenicity when incubated prior to injection with normal serum from several species (Lamoureux et al. 1970, Gerstl et al. 1972, Bernard & Lamoureux 1975a, Pescovitz et al. 1978). The inactivation was earlier ascribed to the binding of MBP by serum proteins, chiefly the a-globulin fraction (Bernard & Lamoureux 1975b). Recently, however, Pescovitz et al. (1978) showed that normal rat serum enzymatically degraded MBP to non-encephalitogenic fragments and that this activity was absent in plasma. We report here the results of our own studies using lz5I-MBP which confirm that MBP is degraded by guinea pig serum. We show that, in addition to losing its encephalitogenicity, serum-treated MBP also loses its ability to suppress EAE and its ability to elicit delayed-type hypersensitivity in MBPsensitized guinea pigs.
0001-6314/79/090140-09 $0.2.50/0 @ 1979 Munksgaard, Copenhagen
141 MATERIAL AND METHODS The isolation and iodination of human MBP were described previously (McDermott et al. 1977). The specific activity of lz5I-MBP was about 3 pCi/pg; this corresponds to a substitution ratio of one iodine atom per 30 molecules of MBP. Blood was removed from guinea pigs by cardiac puncture. Serum was obtained after clotting for 1 h at 37" C, and plasma was obtained using heparin; both were used fresh. Degradation of ' Y - M B P lZSI-MBP(5 X lo6 cpm aprrox.) was added to human MBP (2mg/ml) in phosphate buffered (pH 7.2) saline solution (PBSA). 25 p l of this solution was incubated at 37" C for various times with 50 p l of fresh serum or plasma. The reaction was stopped with 0.5 ml of phenol/formic acid (14:3, w/v). 25 p l of this solution was applied to a horizontal slab gel (12 % polyacrylamide) equilibrated in phenoVformic acid/water (14:3:3, w/v/v) and subjected to electrophoresis at 300 v for 5 h. Gels were fixed in glacial acetic acid, stained with 0.25 % amido black in 7 % acetic acid and destained electrophoretically. Cytochrome-C (12,000 daltons) was used as a marker. The gel was cut into strips which were sliced into 2 mm sections. The radioactivity was determined in a Packard Autogamma counter. Serum treatment of MBP for injection The method of Bernard & Lamoureux (1975a) was used. Human MBP (5 mg) was dissolved in PBSA ( 5 ml); this solution was added to fresh serum (10 ml) and incubated at 37" C for 20 h in a sterile capped tube. Animals Hartley and Strain 13 guinea pigs from our own colonies were maintained on commercial pelleted diet and water ad libitum, with fresh cabbage given three times weekly. Animals were injected with equal amounts of the appropriate emulsion into the dorsum of both hind feet. They were weighed and examined daily for signs of EAE, which were scored as previously (McDermott & Keith 1979). Animals were killed when moribund by exsanguination under ether anaesthesia. Brains and spinal cord were r e moved into formalin (10 %) and histological examination performed on haematoxylin/ eosin stained paraffin-embedded sections as before (Keith 1979). EAE activity MBP in an emulsion of saline (0.1 ml) and FCA (0.1 ml; containing 0.1 mg H37Ra) was injected into Hartley guinea pigs. The serumlMBP mixture from above (0.15 ml) was emulsified with FCA (0.1 ml, containing 0.1 mg H37Ra) and injected into Hartley guinea pigs. Supression of EAE Strain 13 guinea pigs (250-350 g) were injected with 0.2 ml of an emulsion of guinea pig spinal cord homogenate (1:l w/v in saline) with FCA (containing 10 mg/ml H37Ra). Serum-treated MBP was emulsified in incomplete Freund's adjuvant - IFA (1:l v/v) and 0.3 ml injected subcutaneously over the nuchal region at the onset of the first clinical signs of the disease. 0.3 ml of the emulsion was given for a further 9 days (total of 0.5 mg of MBP). In a second treatment group, MBP (50 pg) in 0.1 ml of saline/IFA was given as above. A third group was left untreated.
142 In a second experiment, Strain 13 guinea pigs were challenged with MBP (50 pg) in 0.2 ml of saline/FCA (10 mg/ml H37Ra) and treated with MBP/serum as in the first experiment. Delayed-type skin reactivity Guinea pigs challenged with MBP (50 pg) in FCA were tested at 10 days post-sensitisation for delayed-type hypersensitivity by intradermal injection of 0.1 ml of three saline test solutions: MBP (100 pg); MBP/serum (20 h incubation; 100 pg equivalent), serum (2:1, d v ) . Spot diameters were measured at 48 h.
RESULTS
Degradation of MBP by serum and plasma Initial experiments with serum showed that at 24 h no 1251-MBPremained on the gel. The time course of the degradation was determined by integrating the 1251-MBPpeak for various reaction times. The result (Figure 1) shows that the degradation of MBP is considerably more rapid in serum than in plasma (half life of I2T-MBP: 12 min and 3.8 h, respectively). A second 1251-labelledband was present on the gel in the serum-degraded MBP at '/2 h reaction time (Figure 2a). At 20 h this peak was absent and probably represents an intermediate breakdown product of 1251-MBP.The plasma-degraded 1251-MBPdid not have a prominent peak at this position at any of the reaction times (V2, 4, 8 and 20 h) (Figure 2b). In both the serum and plasma experiments, a small peak of radioactivity was seen at the origin of the gel at all time periods. Loss of encephalitogenic activity None of the animals sensitized with serum-treated MBP in CFA developed clinical or histological signs of EAE (Table 1). Compare this with the high encephalitogenicity of untreated human MBP. Suppression of EAE Untreated Strain 13 guinea pigs had an initial episode of the disease at between 10 and 14 days post-sensitization with guinea pig spinal cord/FCA emulsion. Four animals died in the acute phase of the disease and three animals deteriorated progressively over several weeks. Nine of the 16 animals recovered, but eight of these later suffered relapses. In contrast, the guinea pigs treated with MBP had a milder initial episode in which there were no fatalities. Although six of the eight animals later suffered relapses, the majority of the animals were well when killed at times of 9-45 weeks after sensitization. One animal in the group died after a third relapse at 24 weeks post-sensitization.
143
. 2
4
20
8 Reaction time (hours)
Figure 1 . Rate o f degradation of lZ51-MBPin guinea pig serum and plasma (0-0).
Gel slice number
(0-0)
Gel slice number
Figure 2 . Distribution o f radioactivity ( I z s l ) in polyacrylamide gels after incubation of 1251-MBPwith (a) serum and ( b ) plasma. control; - - - - %-h reaction in serum, 4-h reaction in plasma; -._._20-h reaction.
The guinea pigs treated with serum/MBP resembled the untreated controls in the severity of the initial episode of the disease and also in the overall mortality. Four of the six survivors were killed when clinically well at 4-10
144 Table I . Encephalitogenic activity of serum-treated MBP in Harttey guinea pigs
Challenge
No. developing EAE***
MBP (16,ug)* (32 Pug)* MBP (50 pg)/serum**
* ** ***
5/5 5/5 0/6
In an emulsion of Freund's complete adjuvant (containing lmg/ml H37Ra) and saline (1:l). Human MBP held in fresh guinea pig serum for 20 h at 37" C . Clinical and/or histological evidence for EAE. All the positives had severe clinical signs.
months after sensitization. However, all these animals had moderate to severe histological lesions throughout the CNS. Attempted suppression of acute EAE induced by MBP using the serum/ MBP preparation was completely ineffective, four of the five animals following a rapid and progressive course to death. Delayed-type hypersensitivity All nine tested animals had positive skin reactions to MBP; the mean spot diameter was 10 mm. The serum-treated MBP and serum control injections were negative in all cases. DISCUSSION
The finding that MBP loses its encephalitogenic activity when kept in normal serum at 37" C for several hours agrees with previous reports (Lamoureux et al. 1970, Gerstl et al. 1972, Bernard & Lamoureux 1975a, Pescovtitz et al. 1978). In this case, human MBP and guinea pig serum were used, but the factor responsible for the inactivation of MBP is present in sera from other species (Bernard & Lamoureux 1795a). The loss of encephalitogenicity has been attributed to the binding of serum proteins to MBP which inactivated the encephalitogenic site; cu2-macroglobulin, transferin and ceruloplasmin fractions were found to be the most potent inactivators (Bernard & Lamoureux 1975b). These workers found no evidence for degradation of lZ5I-MBPusing Sephadex G25 column chromatography as the means of identifying small molecular weight (
