Biochemical Changes in Central Nervous System Membranes in Experimental Allergic Encephalomyelitis

Biochemical and morphological studies of myelin subfractions were undertaken on Lewis rats during the early stage of the development of experimental allergic encephalomyelitis (EAE). Myelin subfractions, obtained by sucrose density gradient centrifugation at 10 days post-induction, were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and assayed for 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity. Aliquots were processed for electron microscopic analysis. When comparing the myelin subfractions of EAE-affected animals with those of controls, differences were observed only in the light fractions, i.e., a decrease in the specific activity of CNPase and in the percentage of basic proteins relative to the total proteins of the fraction. This decrease was also evident in the basic protein/proteolipid protein ratio which is frequently used in the literature. In addition, electron microscopic observations demonstrated strong differences in the morphology of the same fraction. These findings suggest that the light fraction is the most sensitive in the early stages of the disease and must play a key role in demyelinating processes.     

A synthetic peptide from myelin proteolipid protein induces experimental allergic encephalomyelitis

Immunization of animals with proteolipid protein, the major protein constituent of central nervous system myelin, produces experimental allergic encephalomyelitis. The goal of the present study was to identify an encephalitogenic determinant of this protein. For this purpose, SWR mice were immunized with five groups of pooled synthetic peptides corresponding to various regions of the myelin proteolipid protein sequence. Clinical EAE was observed in only one group. Inguinal lymph node cells from animals in this group responded ([3H]thymidine incorporation) to a peptide within the pool containing residues 103-116 YKTTICGKGLSATV. Mice subsequently immunized with 50 nmol of this peptide developed severe EAE within 3 wk, and their T cell-enriched inguinal lymph node cells responded specifically to this peptide. Control mice immunized to proteolipid peptide 202-217 DARMYGVLPWNAFPGK did not develop experimental allergic encephalomyelitis, and their inguinal lymph node cells were unresponsive to either peptide. Thus, a peptide corresponding to a sequence within the proteolipid protein can produce classical acute experimental allergic encephalomyelitis. This is the first report of a synthetic encephalitogenic peptide from myelin proteolipid protein.     

Role of lipid interactions in autoimmune demyelination

A morphological transformation involving loss of adhesion between myelin lamellae and formation of myelin vesicles has been described as a mechanism for demyelination in multiple sclerosis and marmoset experimental allergic encephalomyelitis (EAE). Although protein interactions are involved in maintaining normal myelin structure, we describe here how lipids contribute to myelin stability and how lipid changes in EAE, including increases in lipid polyunsaturation and negatively charged phosphatidylserine (PS), promote demyelination. Three physico-chemical techniques were used to identify these changes: (1) Langmuir monolayer isotherms indicated that EAE white matter lipids were significantly more "expanded" (fluid) than controls. (2) NMR spectroscopy indicated that EAE myelin lipids were more polyunsaturated than controls. (3) High-performance liquid chromatography (HPLC) with an evaporative light scattering detector indicated increased PS in EAE compared to controls, while sphingomyelin (SM), sulfatides and phosphatidylcholine (PC) were decreased. We present a physical model considering electrostatic, van der Waals and undulation forces to quantify the effect of these changes on myelin adhesion at the extracellular interface. Taken together, the isotherm, NMR, HPLC and modeling results support a mechanism for autoimmune demyelination whereby the composition of myelin lipids is altered in a manner that increases myelin fluidity, decreases myelin adhesion, increases membrane curvature, and promotes vesiculation.     

Immunomorphological characteristics of experimental allergic encephalomyelitis induced by an encephalitogenic polypeptide

Encephalitogenic, immunogenic properties of the polypeptide fraction of myelin basic protein (FBP) and CNS lesions have been examined in animals with experimental allergic encephalomyelitis (EAE). FBP was isolated from bovine spinal cord using column chromatography. Administration of 1.0 or 0.1 microgram FBP mixed with complete Freund adjuvant caused neurological and histological EAE manifestations in 76% and 26% of guinea-pigs, respectively. Circulating anti-FBP antibodies were not found in sensitized animals, whereas the incidence and intensity of skin reaction of delayed type hypersensitivity to FBP correlated with the development of EAE and the onset of the disease. Perivascular cell infiltration and demyelination noted in the spinal cord and brain of guinea-pigs were similar to those observed after inoculation of the brain white matter or brain tissue homogenate.     

Adoptive transfer of experimental allergic encephalomyelitis in SJL/J mice after in vitro activation of lymph node cells by myelin basic protein: requirement for Lyt 1+ 2- T lymphocytes

Optimal conditions were established for the adoptive transfer of experimental allergic encephalomyelitis (EAE) in SJL/J mice. Lymph node cells from SJL/J mice primed in vivo with myelin basic protein (BP) were incubated in vitro with BP. These cells proliferated specifically to BP and when transferred at the optimal conditions into syngeneic mice induced EAE in 100% of the recipients. The in vitro proliferative response to BP was dependent on the presence of Lyt 1+ 2- T lymphocytes. Furthermore, when the activated LNC were treated before transfer with anti-Thy 1 or anti-Lyt 1 antibody and C, neither clinical nor histologic signs were observed in the recipients, whereas treatment with anti-Lyt 2 antibody and C had no effect. These results indicated that Lyt 1+ 2- T cells are responsible for the transfer of EAE.     

Correlation Between 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase Activity and Demyelination In Vitro Using a Syngeneic System

Cultures of myelinated SJL/J fetal mouse spinal cord were incubated with serum and lymphoid cells from syngeneic animals with experimental allergic encephalomyelitis (EAE) induced by syngeneic spinal cord homogenate (SSCH) in complete Freund's adjuvant or others injected with complete Freund's adjuvant alone. After 24 or 48 h of exposure, demyelination was determined by light microscopic examination and quantification of 2',3'-cyclic nucleotide 3'-phosphohydrolase activity. Cultures exposed to spleen or lymph node cells from SSCH-sensitized animals showed the greatest alterations in myelin and decreases in 2',3'-cyclic nucleotide 3'-phosphohydrolase activity whereas serum from these animals had less effect. Cells and serum from complete Freund's adjuvant-injected control animals also induced structural changes in myelin that were significantly less than changes induced by cells and serum from animals with EAE. These experiments show that lymphoid cells and serum obtained from SJL/J mice with acute EAE affected myelin biochemistry and morphology in syngeneic CNS cultures.     

Mechanism of Myelin Breakdown in Experimental Demyelination: A Putative Role for Calpain

Although calpain has been extensively studied, its physiological function is poorly understood. In contrast, its role in the pathophysiology of various diseases has been implicated, including that of experimental allergic encephalomyelitis (EAE), an animal model of the demyelinating disease multiple sclerosis (MS). In EAE, calpain degrades myelin proteins, including myelin basic protein (MBP), suggesting a role for calpain in the breakdown of myelin in this disease. Subsequent studies revealed increased calpain activity and expression in the glial and inflammatory cells concomitant with loss of axon and myelin proteins. This suggested a crucial role for calpain in demyelinating diseases.     

Suppressor cell control of unresponsiveness to experimental allergic encephalomyelitis.

Lymph node cells (LNC) from Lewis rats rendered unresponsive to experimental allergic encephalomyelitis (EAE) by pretreatment with myelin basic protein markedly suppressed clinical (but not histologic) EAE in normal recipients later challenged with an encephalitogenic emulsion. Unresponsiveness was immunologically specific, and required viable LNC; serum transfer was ineffective. These findings suggest that suppressor cells exert control over this autoimmune disease.     

Correlation between active rosette formation and delayed cutaneous hypersensitivity in experimental allergic encephalomyelitis

The effect of encephalitogenic myelin basic protein, BP, on active rosette-forming T cells (ARFC) was compared to that of nonencephalitogenic peptide S42, a synthetic analogue of the tryptophan region of BP. Depression of ARFC by these antigens was reversible within 24 h after a second dose of the antigen into the skin, or after in vitro incubation of lymphocytes with the sensitizing antigen (Ag-ARFC). The ratio of Ag-ARFC to ARFC rose with time following the sensitization but fell shortly before the clinical onset of experimental allergic encephalomyelitis in animals sensitized with BP. In contrast, the Ag-ARFC/ARFC ratios for animals sensitized with peptide S42 reached plateau levels from which they did not drop. The kinetics of the Ag-ARFC/ARFC responses paralleled those for delayed-type skin hypersensitivity (DTH) in the respective animals. The DTH responses rose following sensitization and fell shortly after the appearance of clinical signs of EAE. The results of this study provide in vitro and in vivo evidence for sensitization to myelin basic protein, and focus attention on the ARFC as a measure for an immunologically active cell population which may be quantitated by antigenic stimulation.Abbreviations used in this report EAE experimental allergic encephalomyelitis - DTH delayed-type skin hypersensitivity - ARFC active rosette-forming T cells - Ag-ARFC antigen-stimulated active rosette-forming T cells - TRFC total rosette-forming T cells     

Active induction of experimental allergic encephalomyelitis

This protocol details a method to actively induce experimental allergic encephalomyelitis (EAE), a widely used animal model for studies of multiple sclerosis. EAE is induced by stimulating T-cell-mediated immunity to myelin antigens. Active induction of EAE is accomplished by immunization with myelin antigens emulsified in adjuvant. This protocol focuses on induction of EAE in mice; however, the same principles apply to EAE induction in other species. EAE in rodents is manifested typically as ascending flaccid paralysis with inflammation targeting the spinal cord. However, more diverse clinical signs can occur in certain strain/antigen combinations in rodents and in other species, reflecting increased inflammation in the brain.     

Myelin metabolism in vitro in experimental allergic encephalomyelitis

Abstract— Spinal cord slices from rats in different stages of allergic encephalomyelitis (EAE) were incubated with [U-¹⁴C]glucose. Normal rats and rats injected with Freund's adjuvant served as controls. The slices were fractionated by a discontinuous sucrose gradient into purified myelin and a heavy membrane residue, the lipids and proteins were extracted, and their specific activities were determined. Uptake of ¹⁴C into myelin lipids was depressed in the rats with acute EAE, while an increase was shown in myelin protein and heavy membrane lipids and proteins. The increased synthesis in non-myelin fractions was ascribed to invasion of metabolically active cells. The depression in myelin lipid synthesis occurred early in the disease before lesions appeared or the inflammatory reaction became widespread. Myelin from guinea pigs with acute EAE resulting from injection of a purified basic protein also showed a depression of uptake in both lipids and proteins. It is suggested that a metabolic insult as a result of the immunological process is dealt the oligodendroglial cells early in the course of the disease which leads to a weakening of the myelin sheath and subsequent phagocytosis of myelin.     

Partial purification and characterization of neutral proteases in lymph nodes of rats with experimental allergic encephalomyelitis

Two kinds of neutral protease activities in lymph nodes from Lewis rats with acute experimental allergic encephalomyelitis (EAE) have been separated and partially purified and characterized. A soluble enzyme preparation enriched by gel filtration and ion-exchange chromatography hydrolyzes myelin basic protein, polylysine, and other basic proteins with an optimum pH at 6.0–6.5. It is inhibited byp-chloromercuribenzoate, and thus appears to be a mixture of thiol proteases. Another fraction containing proteolytic enzyme activity is strongly bound to the insoluble lymph node residue, and it also hydrolyzes myelin basic protein and histone, but not polylysine. It has a pH optimum above 7.5, is inhibited by phenylmethylsulfonyl fluoride, thus resembling elastase, but does not hydrolyze elastin-Congo red. The insoluble enzyme preparation hydrolyzes basic protein to 4–5 peptides in a pattern on polyacrylamide gels resembling that of the hydrolysis of basic protein by whole lymphocytes; the soluble enzyme mixture produces small fragments not retained on gels. Lymphocytes are a major component of the cells inflitrating the nervous system in experimental allergic encephalomyelitis, and neutral proteases contained in these cells may contribute to the degradation of myelin, especially of the basic protein.     

A lymph node neutral proteinase acting on myelin basic protein

A neutral protease present in inguinal and popliteal lymph nodes of rats with acute experimental allergic encephalomyelitis (EAE), rats injected with Freund's adjuvant, and rats that are normal has been found to hydrolyze basic protein present in purified brain and spinal cord myelin. The enzyme has been enriched by ammonium sulfate precipitation, and its properties have been studied. The protease activity toward different substrates was very specific and decreased in the following order: Protamine sulfate = polylysine (MW 183,000) > myelin basic protein > histone > polylysine (MW 2000) > polyarginine > cytochrome c. Other proteins including casein, freshly denatured hemoglobin, egg albumin, bovine serum albumin, and ribonuclease were ineffective as substrates. The pH curve showed a peak at pH7 for rat myelin, isolated beef basic protein, and histone. A possible role for this enzyme in demyelination in acute experimental allergic encephalomyelitis is suggested.     

METABOLIC ACTIVITY OF CNS PROTEINS IN RATS AND MONKEYS WITH EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS (EAE)

—The metabolic activity of proteins from myelin and non-myelin fractions of slices of lesions in monkey brains and in spinal cords of Lewis rats with acute experimental allergic encephalomyelitis was investigated using [1-¹⁴C]leucine as a protein precursor. The uptake in vitro of [1-¹⁴C]leucine into the monkey EAE lesions was greatly increased in both the myelin and non-myelin fractions. Similar findings were made in spinal cord slices of the EAE rat with an average specific activity 341 per cent of control measured in proteins of purified myelin and 415 per cent of control in the non-myelin protein. The increased uptake appeared with the onset of paralytic symptoms 10–14 days after injection. The increased uptake did not appear to be a result of an increased amino acid pool size as measured with uniformly labelled l -leucine, valine, arginine and phenylalanine. The increase in specific activity of the myelin protein of the EAE rats was shown to be associated with the peaks characteristic of myelin protein when separated on polyacrylamide gels and the serial slices counted. Most of the radioactivity of both the control and EAE myelin protein migrated with the high molecular weight fraction, and the largest increase in radioactivity in myelin protein appeared in this fraction. Some increase in specific activity was also found in the basic and proteolipid proteins. Four different guinea-pig antigens were used to induce EAE: whole spinal cord, purified basic protein, purified myelin and basic protein + cerebroside. All caused paralytic symptoms and greatly increased incorporation in vitro of [1-¹⁴C]leucine into spinal cord proteins. The incorporation of [1-¹⁴C]leucine into slices of the inguinal and popliteal lymph nodes of the EAE and Freund's adjuvant control rats were measured and compared with the incorporation into the spinal cord non-myelin fractions. The specific activity of lymph node proteins was of the order of 10 × that of the non-myelin protein of the control spinal cord. Invasion of a moderate number of cells of the order of activity of these lymph nodes could account for the large increase in rate of protein synthesis in the EAE nervous tissue. It is concluded that much of the increased protein synthesis could be due to the inflammatory cells, although a small amount of the total increase appears to be associated with myelin protein. Other changes in metabolism of the CNS tissue of the EAE rat include a lower rate of lipid synthesis and a decreased activity of the tricarboxylic acid cycle.     

Experimental allergic encephalomyelitis-inducing activity of synthetic polyadenylic and polyuridylic homopolymers and complexes in guinea pigs.

In addition to the capacity of polyuridylic acid (poly(U)) or complexes of polyadenylic acid (poly(A)) and Poly(U) (poly(A : U)) to serve as adjuvants for induction of experimental allergic encephalomyelitis (EAE) in guinea pigs sensitized to spinal cord or myelin basic protein, these synthetic polynucleotide homopolymers possess inherent EAE-inducing activity for this host. EAE activity of poly (A) or poly(A : U) was demonstrable following a single injection of the purine homopolymer or the complex in incomplete Freund's adjuvant (IFA). EAE-inducing activity of poly(U) was observed only in guinea pigs initially primed with this pyrimidine homopolymer in IFA.     

Protein kinase Ctheta controls Th1 cells in experimental autoimmune encephalomyelitis

Molecules that regulate encephalitogenic T cells are of interest for multiple sclerosis. In this study we show that protein kinase Ctheta (PKCtheta) is critical for the development of Ag-specific Th1 cells in experimental allergic encephalomyelitis (EAE), a model of multiple sclerosis. PKCtheta-deficient mice immunized with myelin oligodendrocyte glycoprotein failed to develop cell infiltrates and Th1 cytokines in the CNS and were resistant to the development of clinical EAE. CD4 T cells became primed and accumulated in secondary lymphoid organs in the absence of PKCtheta, but had severely diminished IFN-gamma, TNF, and IL-17 production. Increasing Ag exposure and inflammatory conditions failed to induce EAE in PKCtheta-deficient mice, showing a profound defect in the myelin oligodendrocyte glycoprotein-reactive T cell population. These data provide evidence of a pivotal role for PKCtheta in the generation and effector function of autoimmune Th1 cells.     

Chronic experimental allergic encephalomyelitis in guinea pigs: immunologic studies on the two major myelin proteins

Humoral and cell-mediated immunity to the two major myelin proteins, basic protein (MBP) and proteolipid protein, have been investigated during the course of chronic experimental allergic encephalomyelitis (EAE) induced in guinea pigs with whole neural tissue. A positive proliferative response to MBP was observed at 10 and 13 days postimmunization, but was not detectable at subsequent stages. Serum antibodies to MBP first appeared during the chronic stages of the disease. A proliferative response to proteolipid apoprotein was not detected during any stage of chronic EAE. Guinea pigs immunized with proteolipid alone, however, showed a proliferative response. The data suggest that MBP is one of the antigens involved in the induction of the acute episode of chronic EAE, but its role in later stages and that of proteolipid protein remain unknown.     

Histochemistry of oxidative enzymes in experimental allergic encephalomyelitis.

The histoenzymic pattern of oxidative enzymes (G3PD, IDH, SD, G6PD,HBD, NADPH: dehydrogenase) was investigated in experimental allergic encephalomyelitis (EAE) produced in rats according to PATERSON [13]. The results obtained lead to following conclusions: (1) The neuroglia, including the white matter oligodendroglia of immunized rats, exhibits increased oxidoreductase activities; (2) The neuroallergic reaction induces some stimulation of the oxidoreductive metabolism of oligoden-droglia; (3) The enzymatic hyperactivity in EAE does not show any relation to the morphological signs of alterations of the myelin sheath.     

DNA Changes in Spinal Cords of Rats with Experimental Allergic Encephalomyelitis

DNA levels were measured in the spinal cords of Lewis rats during the development of and recovery from experimental allergic encephalomyelitis (EAE). Spinal cord DNA was first increased 11 days after immunizing the rats with guinea pig myelin and rose to levels four times that of the Freund's adjuvant controls at day 14, then subsided after day 22. Spinal cord DNA was still 150% of control levels 60 days after immunization. These DNA changes were compared with fluctuations in spinal cord acid proteinase in the same animals. Acid proteinase activity in EAE spinal cord increased later than the rise in DNA and attained a level of 170% of control at days 15-17, then subsided. Spinal cord DNA was higher in rats immunized with whole myelin than in those administered equivalent amounts of purified myelin basic protein. Furthermore DNA was higher in spinal cords of rats immunized with a larger dose of myelin (1.0 mg) than with a lower amount (0.5 mg). Various protease inhibitors including pepstatin, nitrophenyl p-guanidino benzoate, polylysine, and dipropionyl rhein, previously shown to protect Lewis rats against EAE, suppressed the increase of DNA in the spinal cord. Measurement of DNA increases in the spinal cord of EAE animals provides a convenient reproducible measurement of the severity of inflammation in the CNS and provides an objective criterion for assessment of the efficacy of various agents screened as possible therapeutic treatment for multiple sclerosis.     

Activity of Calpain in the Guinea Pig Brain under Conditions of Experimental Allergic Encephalomyelitis

We studied the activity of calpain in the brain tissue of guinea pigs at different stages of the development of experimental allergic encephalomyelitis (EAE). Eleven days after inoculation of a mixture containing myelin main protein into the experimental animals, we observed a drop in the calpain activity (on average, by 27% with respect to the control), whereas on the 20th and 27th days the activity of the enzyme under study exceeded the norm (by 12%). The calpain/calpastatin ratio also altered at the different stages of development of EAE: the amount of calpastatin increased significantly on the 11th and 27th days, while on the 20th day the level of calpastatin was close to that typical of the control animals. Therefore, we found that the state of calpain/calpastatin system in the guinea pig brain demonstrates some correlation with the dynamics of development of EAE.