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.     

Identification as Synapsin of a Synaptosomal Protein Immunoreacting with Anti-Myelin Basic Protein Antiserum

Rat brain proteins able to react with anti-myelin basic protein antiserum, raised under conditions to induce experimental allergic encephalomyelitis in rabbits, were examined by immunoblot methods after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Apart from the four forms of myelin basic protein present in rat brain, the antiserum detected other proteins of higher molecular weight. Subcellular fractionation shows that these high-molecular-weight proteins are relatively concentrated in a synaptosome-enriched fraction compared to a myelin fraction. A major protein fraction immunorelated to myelin basic protein migrated in the gels as a doublet with apparent molecular weights of approximately 80K and 86K; these proteins were tentatively identified as synapsin Ia and Ib. A purified synapsin preparation analyzed by immunoblot after two-dimensional gel electrophoresis also reacted with anti-myelin basic protein antisera. When the serum was purified by affinity chromatography on a myelin basic protein-conjugated Sepharose column the nonadsorbed material lost this activity whereas the eluted antibodies reacted with myelin basic protein and synapsin. In addition, sequence amino acid comparison of decapeptides showed some homology between these two proteins. A possible implication of immunological agents against myelin basic protein cross-reacting with extra-myelin proteins in the process of experimental allergic encephalomyelitis is considered.     

Enzymic hydrolysis of myelin basic protein and other proteins in central nervous system and lymphoid tissues from normal and demyelinating rats.

Proteolytic activity of central-nervous-system tissue of the normal rat was examined over the pH range 2-9 with casein, haemoglobin and myelin basic protein as substrates. With casein as a substrate, brain and spinal cord homogenates showed very similar activity profiles with increasing pH, with the main peaks of proteolytic activity at pH 3-4 and 5-6. When haemoglobin was used, one broad main peak of activity from pH 3 to 5 was demonstrated. There was no optimum pH, however, for proteolytic activity with myelin basic protein as a substrate, and considerable hydrolysis were observed from pH 3.5 up to pH8. Proteolytic activity at the various pH values was compared by using homogenates of spinal cords from rats with acute experimental allergic encephalomyelitis and those from rats injected with Freund's adjuvant alone. The profiles of activity were similar with peaks at pH 3.5 and 5.5 with casein as a substrate, but the specific activity was significantly higher at most pH values in the spinal-cord homogenates from rats with experimental allergic encephalomyelitis. Similarly the spinal-cord homogenates from these latter rats contained much more proteolytic activity toward myelin basic protein throughout the pH range than was present in the control spinal cords. Homogenates from lymph nodes of rats with experimental allergic encephalomyelitis and from those of the controls contained two to three times as much proteolytic activity as that of the central-nervous-system tissue and had a different proteolytic activity profile form that of the central-nervous system, with higher activity at the neutral than at acid pH. The results are discussed with regard to the probability that inflammatory cells such as lymphocytes may be the cause of the increased proteolytic activity in the central nervous system of animals with experimental allergic encephalomyelitis, and that enzymes from these cells possess the capability of digesting myelin basic protein.     

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.     

Modulation of adoptive cell transfers in experimental allergic encephalomyelitis by antigen treatment of donors

Experimental allergic encephalomyelitis has been adoptively transferred using lymph node cells from Strain 13 guinea pig donors sensitized with purified encephalitogenic myelin basic protein. Adoptive cell transfer was used to examine the immunocompetence of lymph node cells obtained from guinea pigs protected from disease development by treatment with MBP. Lymph node cells from guinea pigs unresponsive to EAE challenge do not adoptively transfer disease. Cells obtained from guinea pigs treated with MBP following encephalitogenic challenge are competent in adoptive transfer with respect to pathologic lesions, but not clinical disease. The clinical and pathologic responses of recipients of the histocompatible lymphocyte populations are similar to those seen in the treatment-matched donor controls, suggesting that under these circumstances lymphoid cells, rather than circulating soluble factors, are responsible for disease induction and suppression.     

PROTEOLYTIC ENZYMES AND EXPERIMENTAL DEMYELINATION IN THE RAT AND MONKEY

Abstract— Visible lesions from monkeys with acute experimental allergic encephalomyelitis (EAE) induced by injection of purified myelin basic protein were assayed for acid proteinase, for a neutral proteinase at pH 6·5, and one lesion was measured for cathepsin A. Acid proteinase was increased to 152–176 per cent of levels in normal-appearing brain areas, neutral proteinase increased to 220–258 per cent, and the one lesion assayed for cathepsin A was 840 per cent of control. These enzymes were measured in the brain stem of Lewis rats with acute EAE as a result of basic protein injection and compared to Freund's adjuvant-injected controls. Acid proteinase was increased significantly to an average level of 128 per cent of control, the increase in neutral proteinase was not significant, and cathepsin A levels were 258 per cent of control, a highly significant increase. The rise in cathepsin A levels was not seen until the onset of paralytic symptoms. The brain stem of Wistar rats treated with whole spinal cord which show EAE in a milder form than the Lewis rat did not contain significantly higher enzyme levels than the control. The increases in acid proteinase and cathepsin A in brain stems were compared to levels of these enzymes in lymph nodes of EAE, Freund's adjuvant-injected controls and uninjected controls. The level of acid proteinase of lymph nodes/g protein did not change appreciably in the course of EAE development in the Lewis and Wistar rats and was about 3–4 times the activity in the brain stem. The cathepsin A in the inguinal lymph nodes of Wistar and Lewis rats injected with whole spinal cord in Freund's adjuvant increases to a level 2× that of the lymph nodes of the uninjected control. The cathepsin A levels in these activated lymph nodes was 6–8 × that of the control brain stem. The lymph nodes of Lewis and Wistar rats injected with Freund's adjuvant alone showed the same increase in cathepsin A as those from rats injected with spinal cord. The brain stem of rats undergoing severe demyelination as a result of chronic administration of triethyl tin did not show the enzyme increases. These results are compatible with the theory that proteolytic enzyme increases in EAE (and probably multiple sclerosis) are due to the invasion of mononuclear cells, some of which are probably lymphocytes. Whether or not these enzymes participate in the actual dissolution of myelin is unknown.     

A sensitive polyacrylamide disc gel method for detection of proteinases

To enable direct detection of proteinase activities subsequent to electrophoresis, a technique utilizing the incorporation or diffusion of protein substrates into polyacrylamide disc gels was developed. Denatured insoluble substrates, casein or hemoglobin, were added to acrylamide solutions prior to polymerization of the gel mixture. Alternatively, soluble protein substrates were diffused into gels after electrophoresis. In either case, an incubation period ensued at the pH optimum of the proteinases to allow for their detection. Classification of resolved proteinases was accomplished subsequent to electrophoresis by incubation of gels in media containing either synthetic substrates, as the naphthylamide derivatives, or specific inhibitors of the enzymes. Separation of purified trypsin from chymotrypsin, and proteinases in preparations of seminal plasma and mouse blastocysts homogenates demonstrated the efficacy of the method at the submicrogram enzyme level.     

Identification of Elastase as a Secretory Protease from Cultured Rat Microglia

In the course of studying the secretory products of microglia, we detected protease activity in the conditioned medium. Various proteins (casein, histone, myelin basic protein, and extracellular matrix) were digested. The protease activity was characterized by using purified myelin basic protein as a substrate. Maximal activity was observed at neutral pH levels (7-8), which was different from the optimum pH level of proteolytic activity observed in the cell homogenate. The activity was inhibited approximately 60 and 50% by 1 mM phenylmethylsulfonyl fluoride and 40 microM elastatinal, respectively. In gel filtration, the major activity, which was inhibited in the presence of N-methoxysuccinyl-Ala-Ala-Pro-Val-methyl chloride, eluted at a position corresponding to a molecular mass of approximately 25 kDa. These results suggest that the major protease present in microglial conditioned medium is elastase or an elastase-like protease. This suggestion was confirmed by the finding that the 25-kDa protein band was stained with anti-elastase antiserum by western blotting. De novo synthesis of elastase in microglia was supported by [35S]methionine incorporation. In the presence of lipopolysaccharide, the secretory elastase decreased. These results demonstrate that microglia secrete proteases, one of which was identified as elastase. The significance of this enzyme production in physiological and pathological conditions is discussed.     

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.     

The breakdown of myelin-bound proteins by intra- and extracellular proteases

Changes in protein components of purified myelin were measured following incubation in vitro with purified intra- and extracellular enzymes. Incubation with calf brain cathepsin D did not result in a significant relese of acid-soluble peptides as measured by ninhydrin analysis but was accompanied by a large loss of myelin proteins as determined on SDS-acrylamide gels. After 5 hr at 37°C there was a loss of about 25% for fast and slow basic proteins and the Agrawal proteolipid, but only a 5–10% loss for the Folch-Lees and Wolfgram components. Rat brain cathepsin D prepared by affinity chromatography gave a 30–60% breakdown of basic proteins and proteolipids. In general, breakdown using lyophilized myelin was increased over two-fold as compared to experiments with fresh myelin. Breakdown induced by cathepsin D was completely inhibited by the pentapeptide pepstatin. Incubation of myelin at physiological pH resulted in an endogenous breakdown of about 12% for basic proteins in freshly prepared, and 50% for lyophilized material. Addition of a soluble neutral proteinase that splits hemoglobin did not induce additional breakdown except for a small change in the Folch-Lees component. The extracellular enzymes pepsin and TPCK-treated trypsin resulted in a larger breakdown of all components as compared to brain enzymes. Present results demonstrate that all protein components of myelin are accessible to hydrolases and vulnerable to breakdown to varying extents by brain enzymes. These facts are consistent with the known rates for myelin protein turnover and may have a bearing on changes associated with demyelinating diseases     

The development of experimental allergic encephalomyelitis with immunizing doses of myelin basic protein.

Rabbits immunized with low (11.25 mg) and high (57.50 mg) doses of myelin basic protein from several species develop antibasic protein antibodies, delayed type hypersensitivity, and clinical and pathological signs of experimental allergic encephalomyelitis. More than 55% of rabbits immunized with relatively high doses of basic protein develop disease. The absence of circulating antibasic protein antibodies in immunorespondent animals is associated with the appearance of clinical or histological signs of experimental allergic encephalomyelitis; however, the presence of humoral antibodies did not prevent completely the development of disease. Delayed-type hypersensitivity, specific for the basic protein, appears as early as 5 days after immunization and is maintained in nondiseased and surviving animals. Neither excess encephalitogen nor encephalitogen-induced antibody is sufficient to suppress completely the eventual development of clinical or histological manifestations of disease.     

Photolabeling of myelin basic protein in lipid vesicles with the hydrophobic reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine

The hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine([125I]TID) was used to label myelin basic protein or polylysine in aqueous solution and bound to lipid vesicles of different composition. Although myelin basic protein is a water soluble protein which binds electrostatically only to acidic lipids, unlike polylysine it has several short hydrophobic regions. Myelin basic protein was labeled to a significant extent by TID when in aqueous solution indicating that it has a hydrophobic site which can bind the reagent. However, myelin basic protein was labeled 2-4-times more when bound to the acidic lipids phosphatidylglycerol, phosphatidylserine, phosphatidic acid, and cerebroside sulfate than when bound to phosphatidylethanolamine, or when in solution in the presence of phosphatidylcholine vesicles. It was labeled 5-7-times more than polylysine bound to acidic lipids. These results suggest that when myelin basic protein is bound to acidic lipids, it is labeled from the lipid bilayer rather than from the aqueous phase. However, this conclusion is not unequivocal because of the possibility of changes in the protein conformation or degree of aggregation upon binding to lipid. Within this limitation the results are consistent with, but do not prove, the concept that some of its hydrophobic residues penetrate partway into the lipid bilayer. However, it is likely that most of the protein is on the surface of the bilayer with its basic residues bound electrostatically to the lipid head groups.     

In vivo immunomodulation by monoclonal anti-CD4 antibody. II. Effect on T cell response to myelin basic protein and experimental allergic encephalomyelitis

In vivo administration of anti-CD4 mAb (GK1.5) has been shown to be effective in preventing acute and relapsing experimental allergic encephalomyelitis (EAE). In the present report we have studied the depletion of CD4+ cells by a single dose of GK1.5 on the immune response to myelin basic protein and in the development of EAE. Our studies show that depletion of CD4 cells in mice that had received encephalitogenic CD4+ T cells altered the kinetics of acute and relapsing EAE, but did not prevent disease altogether. The in vitro T cell proliferative response to myelin basic protein in lymph node cells was maintained in the presence of significant depletion of CD4+ cells. These studies indicate that the population of Ag-reactive cells to be large and relatively refractory to antibody therapy. The implication of these results to therapy of human autoimmune disease is discussed.     

Soluble bovine galactose-binding lectin. cDNA cloning reveals the complete amino acid sequence and an antigenic relationship with the major encephalitogenic domain of myelin basic protein.

A full-length cDNA clone for the 13-14 kDa soluble beta-galactoside-binding lectin was isolated from a bovine fibroblast cDNA library. The derived amino acid sequence shows eight differences from a preliminary partial amino acid sequence given previously for the bovine heart lectin. This observation led to a re-examination of the data and correction of the heart lectin protein sequence. Except for a possible polymorphism of the heart lectin at position 57, the fibroblast and heart lectin sequences are considered identical. The epitope recognized by two monoclonal anti-(bovine lectin) antibodies, 36/8 and 9/5, was identified as the tetrapeptide sequence W-G-A/S-E/D by the isolation of several different cDNA clones from a human intestine cDNA library. A similar tetrapeptide is present in all of the soluble beta-galactoside-binding animal lectins sequenced thus far. It is also found in myelin basic protein, which we show is antigenically cross-reactive with the lectin. In myelin basic protein the tetrapeptide is a part of the major domain previously shown to be responsible for the induction of experimental allergic encephalomyelitis.     

Effect of poly-basic amino acids on the phosphorylation of various substrate proteins by cytosolic protein-tyrosine kinase from porcine spleen

Cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) is strongly activated by poly-L-lysine using bovine serum albumin, ovalbumin, phosphorylase b, calmodulin and H1 histone as substrate proteins. However, this polyamine inhibited the enzyme activities when myelin basic protein, tubulin and H2B histone were used as substrate proteins. These stimulatory and inhibitory effects on CPTK-40 are not specific for polylysine, but polyarginine and polyornithine have similar effects on this phosphorylation reaction. Effect of poly-basic amino acids on CPTK-40 seems to be mainly on the substrate proteins, rather than on the enzyme itself.     

Cytotoxicity of sera against brain and spinal cord antigens in relation to lymphocytes of varying origin]

Rabbit sera against antigens prepared from the brain and the spinal cord antigens were investigated in a cytotoxic test with mouse and guinea pig lymphocytes. None of the sera exerted a cytotoxic effect on the bone marrow lymphocytes. The sera against mouse brain and spinal cord and guinea pig brain and myelin isolated from it exerted the greatest cytotoxic activity; the cytotoxicity was maximum against the thymocytes, less pronounced against the lymph node lymphocytes, and least--against the spleen cells. The cytotoxicity of the sera against the bovine spinal cord homogenate, myelin and the basic protein isolated from it was the minimal and equal with lymphocytes from any of the three mentioned sources. The serum against the encephalitogenic polypeptide 2c was practically devoid of the cytotoxic activity. The encephalitogenic activity of the 2c fraction was greater than that of the myelin and basic protein from the bovine spinal cord. Experiment of antibrain serum absorption suggested that the brain cortex contained a cross-reacting antigen. The subcutaneous injection of a relatively high dose (224 x 10(6)) of thymocytes in the complete Freund's adjuvant failed to induce the development of allergic encephalomyelitis in guinea pigs.     

PHOSPHOPROTEIN PHOSPHATASE OF PINEAL GLAND: SOME PROPERTIES OF THE ENZYME AND THE IDENTIFICATION OF AN ENDOGENOUS ACTIVATOR

Abstract— Both soluble and insoluble fractions of rat pineal glands catalyze the dephosphorylation of phosphohistone. The phosphoprotein phosphatase in cytosol as well as in insoluble fraction is inhibited by ZnCl₂ and NaF. Guanosine triphosphate, ATP and MnCl₂ activate the soluble enzyme but not the enzyme in the insoluble fraction suggesting that with solubilization from membranes some unfunctional changes of the enzyme may occur. Fractionation of the soluble enzyme preparation revealed the existence of two forms of enzyme differing in molecular weight. These two forms can be further differentiated by their sensitivities to MnCl₂ and deoxycholate. A thermostable factor which activates the soluble but not the insoluble enzyme was demonstrated in both beef and rat pineal glands. The thermostable factor is protein in nature because it is nondialyzable and trypsin labile. Whether in vivo the endogenous activator mediates the regulation of the phosphoprotein phosphatase in pineal remains to be investigated.     

Neonatal exposure to antigen primes the immune system to develop responses in various lymphoid organs and promotes bystander regulation of diverse T cell specificities

Neonatal exposure to Ag has always been considered suppressive for immunity. Recent investigations, however, indicated that the neonatal immune system could be guided to develop immunity. For instance, delivery of a proteolipid protein (PLP) peptide on Ig boosts the neonatal immune system to develop responses upon challenge with the PLP peptide later. Accordingly, mice given Ig-PLP at birth and challenged with the PLP peptide as adults developed proliferative T cells in the lymph node that produced IL-4 instead of the usual Th1 cytokines. However, the spleen was unresponsive unless IL-12 was provided. Herein, we wished to determine whether such a neonatal response is intrinsic to the PLP peptide or could develop with an unrelated myelin peptide as well as whether the T cell deviation is able to confer resistance to autoimmunity involving diverse T cell specificities. Accordingly, the amino acid sequence 87-99 of myelin basic protein was expressed on the same Ig backbone, and the resulting Ig-myelin basic protein chimera was tested for induction of neonatal immunity and protection against experimental allergic encephalomyelitis. Surprisingly, the results indicated that immunity developed in the lymph node and spleen, with deviation of T cells occurring in both organs. More striking, the splenic T cells produced IL-10 in addition to IL-4, providing an environment that facilitated bystander deviation of responses to unrelated epitopes and promoted protection against experimental allergic encephalomyelitis involving diverse T cell specificities. Thus, neonatal exposure to Ag can prime responses in various organs and sustain regulatory functions effective against diverse autoreactive T cells.     

The interaction of myelin basic protein with tubulin and the inhibition of tubulin carboxypeptidase activity

Tubulin carboxypeptidase was found to be inhibited by myelin basic protein in a concentration dependent manner. The inhibition was produced by the interaction between myelin basic protein with the substrate. As a consequence of this interaction, turbid insoluble aggregates were formed at either 5 degrees or 37 degrees C. The turbidity increased by increasing the myelin basic protein concentration and it reached a plateau at a molar ratio of myelin basic protein to tubulin dimer of about 6. At plateau, the molar ration in the insoluble aggregates was about 6. When tubulin was in excess, the formation of the insoluble aggregates was diminished. However, if the excess of tubulin was added after the formation of the aggregates, the turbidity was not significantly affected. Turbidity was diminished by increasing the ionic strength.     

Dissociation of experimental allergic encephalomyelitis protective effect and allergic side reactions in tolerization with neuroantigen

Administration of autoantigens under conditions that induce type 2 immunity frequently leads to protection from T cell-mediated autoimmune diseases. Such treatments, however, are inherently linked to the induction of IgG1 Abs and to the risk of triggering anaphylactic reactions. We studied the therapeutic benefit vs risk of immune deviation in experimental allergic encephalomyelitis of SJL mice induced by MP4, a myelin basic protein-proteolipid protein (PLP) fusion protein. MP4 administration in IFA induced type 2 T cell immunity, IgG1 Abs, and experimental allergic encephalomyelitis protection, and all three were enhanced by repeat injections. Despite high Ab titers, anaphylactic side reactions were not observed when MP4 was repeatedly injected in IFA or as soluble Ag s.c. In contrast, lethal anaphylaxis was seen after s.c. injection of soluble PLP:139-151 peptide, but not when the peptide was reinjected in IFA. Therefore, the Ab response accompanying the immune therapy constituted an anaphylactic risk factor only when the autoantigen was not retained in an adjuvant and when it was small enough to be readily disseminated within the body. Taken together, our data show that treatment regimens can be designed to boost the protective type 2 T cell response while avoiding the risk of Ab-mediated allergic side effects.