Redistribution of Lyt-bearing T cells in acute murine experimental allergic encephalomyelitis: selective migration of Lyt-1 cells to the central nervous system is associated with a transient depletion of Lyt-1 cells in peripheral blood

Experimental allergic encephalomyelitis (EAE) was induced in SJL/J mice by using two injections of spinal cord homogenate in incomplete Freund's adjuvant supplemented with mycobacteria. Analysis of circulating Lyt-bearing subsets by indirect immunofluorescence during the course of acute EAE revealed the following: 1) during the pre-clinical phase of EAE (1 to 2 days before the onset of paralysis), there was a decrease in the percentage of Lyt-1- but not of Lyt-2-bearing cells in peripheral blood, and of both Lyt-1- and Lyt-2-bearing cells in spleen; 2) with the onset of clinically evident EAE, there was a decrease in both Lyt-1 and Lyt-2 cells in peripheral blood and an increase in the percentage of Lyt-1-bearing cells in pooled inguinal and axillary lymph node; and 3) after these early changes, there was a rapid reconstitution of the percentages of total Lyt-bearing cells and of both Lyt-1- and Lyt-2-bearing cells in peripheral blood. Immunohistochemical analysis of the central nervous system infiltrate revealed that the earliest lesions consisted predominantly of Lyt-1 T lymphocytes, with few Lyt-2 cells present. These results demonstrate that the influx of cells of the Lyt-1 inducer subset to the central nervous system in acute EAE is accompanied by a transient decrease in Lyt-1 cells in peripheral blood.     

Relapsing murine experimental allergic encephalomyelitis induced by myelin basic protein

Experimental allergic encephalomyelitis (EAE), an experimental autoimmune disease of the central nervous system (CNS), is readily induced in many mammalian species by immunization with CNS tissue or myelin basic protein (MBP) purified from the CNS. EAE has been frequently used as a model for multiple sclerosis (MS). However, EAE generally presents as an acute monophasic disease in the adult animal after immunization with MBP. After recovery, the animal is resistant to rechallenge with encephalitogen (1). Two exceptions to these observations have been reported. McFarlin et al. (2) reported that a variable number of Lewis rats showed signs of a single, mild relapse about a week after recovery from MBP-induced acute EAE. Panitch and Ciccone (3) have reported induction of recurrent EAE in rats immunized with human MBP. Chronic, relapsing EAE has been induced in the mouse; however, an apparent requirement for CNS tissue had been noted (4, 5). Recently, during the course of a series of experiments on the induction of EAE in SJL/J, PL/J, and (SJL/J X PL/J)F1 (SPL F1) mice, it was observed that the F1 mice frequently had paralytic relapses after recovery from MBP-induced symptoms. Experiments were initiated to examine this phenomenon, and the findings are presented below.     

Specifically reactive cells in experimental allergic pertussis encephalomyelitis]

Dynamics of emergence of specific reactive cell (SRC) with respect to the brain antigen in the draining regional lymph nodes and peripheral blood was studied in experimental whooping cough allergic encephalomyelitis (EAE) in guinea pigs. The greatest number of SRC in the regional lymph nodes, that markedly decreased by the 9th day of sensitization, was revealed in the middle of the EAE incubation period (the 6-7th day), whereas the peripheral blood showed the highest SRC number during this period. The SRC number rose in the regional lymph nodes and dropped in the peripheral blood at the height of EAE progress (the 20th day). It is concluded that SRC found may be attributed to T lymphocyte population.     

Lipid peroxidation in experimental allergic encephalomyelitis

Lipid peroxidation (LPO) in the brain and blood of guinea-pigs was studied during experimental allergic encephalomyelitis. The most pronounced activation of LPO in the brain occurred at the 7th day of sensitization with encephalolitogenic emulsion. It manifested by an increase in the content of diene conjugates and malonic dialdehyde, activation of catalase and reduction of superoxide dismutase activity. LPO activation in the blood occurred at the 3th-5th day of sensitization. It is assumed that LPO activation is caused by antigen-antibody reaction that occurs in the blood at the 3d day and in the brain at the 7th day of sensitization.     

Passive induction of experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is a widely used animal model of the human demyelinating disease multiple sclerosis. EAE is initiated by immunization with myelin antigens in adjuvant or by adoptive transfer of myelin-specific T cells, resulting in inflammatory infiltrates and demyelination in the central nervous system. Induction of EAE in rodents typically results in ascending flaccid paralysis with inflammation primarily targeting the spinal cord. This protocol describes passive induction of EAE by adoptive transfer of T cells isolated from mice primed with myelin antigens into na?ve mice. The advantages of using this method versus active induction of EAE are discussed.     

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.     

The role of mast cells in the elicitation of experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis (EAE), a T cell-mediated autoimmune disease can be transferred with lymphoid cells from actively immunized rats into naive recipients. In the mouse, previous studies have suggested a role for histamine/serotonin in the development of active EAE. We have found that myelin basic protein-reactive cells transfer a biphasic skin test response to naive rats analogous to what has been described in the mouse contact dermatitis system, where mast cell sensitization by Ag-specific T cell factors is required for the induction of skin test responses. Treatment of cell recipients with the serotonin receptor antagonists, cyproheptadine or methysergide, blocked or significantly reduced the development of EAE. Furthermore, it was found that treatment with cyproheptadine was effective in blocking clinical disease when administered day 3 to day 6 after cell transfer. In contrast, cyproheptadine treatments before induction of paralysis day 0 to 3, failed to alter the course of clinical disease. The inhibitor of mast cell degranulation, proxicromil, was also found to effectively block the elicitation of adoptively transferred EAE and was also found to be effective when administered just before the onset of clinical disease. Reserpine, a compound known to deplete mast cells of vasoactive amines by forcing granule contents into the cytoplasm where they are degraded by cell enzymes, was also effective in blocking both active and adoptively transferred EAE. Disease inhibition was found to be partially reversed with pargyline, an inhibitor of monoamine oxidase. In addition lymphocytes from treated animals were capable of transferring disease to naive recipients and appeared to have normal activity as assessed by Ag-or mitogen-driven proliferation in addition to IL-2 production.     

Neutral protease activity in lymphocytes of lewis rats with acute experimental allergic encephalomyelitis

Lymphocytes from popliteal and inguinal lymph nodes of Lewis rats with acute EAE as a result of injection of lyophilized guinea pig myelin in Freund's complete adjuvant exerted strong proteolytic activity at neutral pH toward myelin basic protein. After injection of myelin the level of proteolytic activity remained about the same as that in lymphocytes from Freund's adjuvant-injected controls until about day 10 after injection, just before the onset of paralytic symptoms; then the proteolytic activity increased to approximately double its former level. Myelin basic protein was hydrolyzed by whole lymphocytes, but more activity was unmasked by homogenization. Similar results were also obtained using lymphocytes from thymus of EAE and control animals. Lymphocytes with high levels of proteolytic activity were not absorbed by glass wool, did not stain with neutral red, nor did they phagocytose antibody-coated sheep red blood cells. Thymus and lymph node lymphocytes cleaved myelin basic protein to three major peptides and a fourth minor peptide, while spleen lymphocytes hydrolyzed basic protein at only one point resulting in two peptides whose molecular weights added up to that of myelin basic protein. The protease activity was inhibited by 5×10^–3 Mp-chloromercuribenzoate and by phenylmethyl sulfonyl fluoride, TPCK, and soybean trypsin inhibitor, therefore the enzymatic activity probably depends on a serine residue and a sulfhydryl group. The bulk of the enzymatic activity is mostly membrane bound with the highest specific activity and total activity contained in a lysosomal-mitochondrial fraction. In view of the infiltration of lymphocytes into the brain substance in acute EAE, it is suggested that these cells may contribute to the destruction of myelin which is usually attributed to the monocyte or macrophage.     

Successful treatment of experimental allergic encephalomyelitis with transforming growth factor-beta 1.

Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional cytokine with immunosuppressive effects on T cells in vitro. Experimental allergic encephalomyelitis is an archetypal T cell-mediated autoimmune demyelinating disease of the central nervous system that often serves as a model for multiple sclerosis. In vivo administration of TGF-beta 1 into SJL mice was successful in reducing the incidence of clinical disease and the histologic severity of inflammation and demyelination in the brain and spinal cord. Immunohistochemical studies performed on control animals showed that TGF-beta-1, -2, and -3 were present in inflammatory perivascular lesions in the brain. The use of a naturally occurring cytokine with immunoregulatory functions in the treatment of an autoimmune disease is novel. However, potential long term complications of such therapy must be addressed before its use in human autoimmune disease such as multiple sclerosis.     

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.     

Activation of regulatory cells suppresses experimental allergic encephalomyelitis via secretion of IL-10

Suppression of CD4+ Th1 cell-mediated autoimmune disease via immune deviation is an attractive potential therapeutic approach. CD4+ Th2 T cells specific for myelin basic protein, induced by immunization of young adult male SJL mice, suppress or modify the progression of CNS autoimmune disease. This report demonstrates that activation of non-neuroantigen-specific Th2 cells is sufficient to suppress both clinical and histological experimental allergic encephalomyelitis (EAE). Th2 cells were obtained following immunization of male SJL mice with keyhole limpet hemocyanin. Transfer of these cells did not modify EAE, a model of human multiple sclerosis, in the absence of cognate Ag. Disease suppression was obtained following adoptive transfer and subcutaneous immunization. Suppression was not due to the deletion of myelin basic protein-specific T cells, but resulted from the presence of IL-10 as demonstrated by the inhibition of Th2-mediated EAE suppression via passive transfer with either anti-IL-10 or anti-IL-10R mAb. These data demonstrate that peripheral activation of a CD4+ Th2 population specific for an Ag not expressed in the CNS modifies CNS autoimmune disease via IL-10. These data suggest that either peripheral activation or direct administration of IL-10 may be of benefit in treating Th1-mediated autoimmune diseases.     

Correlation of cytotoxicity with experimental allergic encephalomyelitis.

Cytotoxicity of immune lymph node cells in experimental allergic encephalomyelitis (EAE) was maximal 9 days after injection of encephalitogenic emulsion. The ability of these cells to passively transfer EAE was also maximal at this time. Immune spleen cells were more cytotoxic than lymph node cells 9 days after injection; however, these cells did not passively transfer EAE. Twelve days after injection of encephalitogenic emulsion immune spleen cells passively transferred EAE with resulting mild histopathologic lesions. At this time the spleen cells were 50% more cytotoxic than comparable lymph node cells. Cyclophosphamide suppressed the development of clinical EAE and the development of cytotoxic lymphoid cells. It also reduced clinical signs and cytotoxic activity of lymph node cells. Spleen cell cytotoxic activity was enhanced by Cyclophosphamide. It was concluded that cytotoxic activity of lymph node and spleen cells was correlated with the ability of these cells to produce EAE. Lymph node cell populations differed qualitatively and/or quantitatively from immune spleen cell populations in EAE. Capacity to passively transfer EAE coincided with the maximal Cytotoxicity of the lymphoid cells from each tissue.     

Therapy of murine leukemia with cyclophosphamide and immune Lyt-2+ cells: cytolytic T cells can mediate eradication of disseminated leukemia

Several animal models have been developed in which the adoptive transfer of specifically immune syngeneic T cells has been shown to mediate the eradication of established tumors. In adoptive chemoimmunotherapy (ACIT) of disseminated FBL leukemia with cyclophosphamide and immune T cells, the major effector T cell has been shown to be a noncytolytic Lyt-1+2- T cell that mediates its therapeutic effect without the participation of CTL. Because studies in other models have suggested that CTL can mediate an anti-tumor effect, the efficacy of Lyt-2+ T cells rendered highly cytolytic before adoptive transfer in ACIT of disseminated FBL was examined. The results demonstrated that such CTL had a detectable but limited therapeutic effect in the treatment of FBL. Because this limited activity of transferred purified CTL might have reflected a requirement for helper T cells to produce IL 2 for promotion of the in vivo survival and proliferation of the CTL, the effect of administering IL 2 to tumor-bearing hosts after transfer of CTL was examined. A dose of IL 2 previously shown to induce in vivo proliferation of transferred T cells rendered CTL that were minimally effective alone curative in ACIT of FBL leukemia. Thus, either lymphokine-producing T cells or the lymphokines produced by these cells are necessary for the full expression of the in vivo therapeutic potential of CTL.     

Immunopathology of murine experimental allergic orchitis

Experimental allergic orchitis (EAO) was induced consistently in BALB/c mice by immunization with homologous testicular tissue homogenate emulsified in complete Freund's adjuvant (CFA) providing that the animals had received simultaneously at least 1 microgram of an extract of Bordetella pertussis rich in pertussigen. All animals thus treated developed orchitis and serum antibody to testicular antigens within 20 days after immunization. The lesions were located in testis (100%), rete testis (37%), cauda epididymis (21%), and vas deferens (37%). Ductus efferentes and caput epididymis were only rarely affected. Early lesions in the seminiferous tubules were characterized by peritubular and/or intratubular accumulation of eosinophils, neutrophils, lymphocytes, and macrophages. This was followed by aspermatogenesis. Late lesions included massive necrosis and extensive fibrosis of the seminiferous tubules. Disruption of blood-testis barrier on day 20 was evidenced by the detection of 1) perfused lanthanum deposits between Sertoli cells and surrounding inflammatory cells inside the seminiferous tubules, 2) deposits of endogenous mouse IgG in germinal epithelium, and 3) probable immune complexes (granular C3) surrounding seminiferous tubules. Murine EAO differed from that of the guinea pig in the lack of involvement of the ductus efferentes, the extensive necrosis, the abundant polymorphonuclear eosinophils in the lesion, and the exquisite requirement of concomitant injection of B. pertussis extract.