Clinical Experimental Allergic Encephalomyelitis Research Articles

In situ detection of class I and II major histocompatibility complex antigens in the rat central nervous system during experimental allergic encephalomyelitis: An immunohistochemical study

To determine in situ localization of cells bearing major histocompatibility complex (MHC) class I or II antigens in the central nervous system (CNS), immunohistochemical examination was performed on CNS sections of Lewis rats sensitized for experimental allergic encephalomyelitis (EAE). Class I antigens identified by OX18 were detected on endothelial cells (EC) and cells with dendritic morphology (DC) of normal rats. OX18 + DC increased in number as the clinical signs of EAE became more severe, while the number of OX18 + EC in clinical EAE rats was not different from that of normal control rats. Infiltrating lymphocytes were always observed around OX18 + vessels. Double staining showed that OX18 + DC was negative for glial fibrillary acidic protein (GFAP). Cells with morphological features of oligodendroglia were not detected with OX18 in both normal control and EAE rats. MHC class II antigens (Ia antigens) were detected using three MAbs: OX3, OX6 and OX17. These three different MAbs essentially showed the same staining pattern. In normal controls, mononuclear cells in the subarachnoid space were stained positively, but no Ia + parenchymal cells were detected. In EAE rats, Ia + DC were first detectable in the white matter of the spinal cord at the preclinical stage, and increased in number as the disease progressed. On the other hand, double-staining with OX6 and anti-factor VIII-related antigen antiserum, or with OX3 and anti-vimentin antiserum demonstrated that endothelial cells even with lymphocyte cuffing were negative for Ia antigens. Based on the data obtained in the present study, the possible role of MHC class I and II antigens in the development of EAE is discussed.

Experimental allergic encephalomyelitis induced by proteolipid apoprotein in Lewis rats

Experimental allergic encephalomyelitis (EAE) was induced in inbred Lewis rats by sensitization with bovine white matter proteolipid apoprotein (PLP). 18–61 days after a single injection of 100 μg of PLP, 12 of 31 rats (39%) developed clinical EAE and 18 of 23 (78%) showed pathologic EAE with significant demyelination. Lymphocyte proliferative responses and antibodies to PLP were elevated but did not correlate with the clinical or pathologic state. This is the first demonstration of PLP-induced EAE with significant demyelination in rats and will contribute to the study of autoimmune demyelination.

Immunohistochemical analysis of the rat central nervous system during experimental allergic encephalomyelitis, with special reference to Ia-positive cells with dendritic morphology.

The rat central nervous system (CNS) during experimental allergic encephalomyelitis (EAE) was analyzed immunohistochemically from the preclinical to recovery stage by using monoclonal antibodies specific for rat T lymphocyte subsets and Ia antigen. Through combination of the avidin-biotin technique and carefully selected fixative, cells with dendritic morphology (DC) and infiltrating mononuclear cells were clearly and intensely demonstrated in the CNS parenchyma during EAE. In normal and complete Freund's adjuvant (CFA)-injected controls, there were no inflammatory foci. Ia (OX3)-positive parenchymal cells were not detected, whereas W3/25 stained DC that were located mainly in the white matter and W3/13 stained axons. At the preclinical stage, 11 days after CNS/CFA sensitization, a few clusters of Ia+ DC were detected in some sections of the spinal cord. The number of Ia+ DC increased as clinical signs developed (P less than 0.001). In rats with a clinical score of 1 or 2, Ia+ DC were mainly located in the perivascular region and closely associated with infiltrating T lymphocytes. However, at moribund state (score 3), Ia+ DC were evenly distributed in gray and white matter on almost all sections of the spinal cord. In recovered rats, the numbers of inflammatory foci and Ia+ DC were less than those in clinical EAE rats (P less than 0.001). Rats without clinical signs throughout the course also contained a few clusters of Ia+ DC. Double immunofluorescent staining with OX3 and anti-glial fibrillary acidic protein (GFAP) antiserum demonstrated that Ia+ DC were negative for GFAP. Their morphology and distribution were similar to those of nucleoside diphosphatase-positive cells, suggesting that Ia+ DC are microglia. In contrast to DC, no astrocytes or endothelial cells express detectable levels of Ia antigen in control and clinical EAE rats. These findings suggest that brain cells other than Ia+ DC may not be involved in the local immune interaction. Ia+ DC may play a significant role in antigen presentation in the CNS with EAE.

Characterization of T cell lines and clones from SJL/J and (BALB/c x SJL/J)F1 mice specific for myelin basic protein.

Lines of thymus-derived lymphocytes reactive against bovine myelin basic protein (BP) were established in vitro from SJL/J mice. These lines are stable in long-term culture and mediate inflammatory central nervous system (CNS) lesions and a low incidence of clinical experimental allergic encephalomyelitis (EAE) when injected into recipient SJL/J mice. The line cells proliferate in response to BP of bovine, rat, or mouse origin. Clones were derived from these lines, and the characteristics of these clones were analyzed. The clones express Thy-1, Ly-1, and L3T4 antigens and are negative for Ly-T2. The clones all proliferate in response to bovine BP, with different clones showing varying degrees of cross-reactivity between bovine, rat, and mouse BP. The proliferative response is MHC-restricted; antigen-presenting cells from I-As strains are required. Compatible with their phenotype as helper cells, some of the clones will provide help to primed B cells stimulating antibody production in an in vitro assay. When injected into recipients pretreated with pertussis and irradiation, clones that showed proliferation to mouse BP induced the development of inflammatory lesions in the CNS, with mortality of 28% of the recipients. T cell lines were also established in (BALB/c x SJL/J)F1 mice. In contrast to the homozygous SJL/J lines, these lines were highly encephalitogenic, inducing a high incidence of clinical and histologic EAE when injected in vivo.

The nature of the defect in experimental allergic encephalomyelitis (EAE)-resistant Lewis (Le-R) rats.

Recently, a colony of Lewis rats has been described which is resistant to experimental allergic encephalomyelitis (EAE). These rats, termed Le-R, are still histocompatible with other Lewis rats. The genetic defect which results in EAE-resistance was shown not to be linked to the RT1.B (Ir) region of the MHC. Myelin basic protein (BP)-sensitization of Le-R rats induces cells capable of mounting a proliferative response to BP in culture but incapable of transferring EAE after culture with BP. The present study demonstrates that the latter deficiency can be overcome either by incorporating lipopolysaccharide (LPS) in the BP-culture medium or by simultaneous transfer of LPS-activated antigen-nonspecific spleen cells with the BP-sensitized cells. The BP-sensitized Le-R cells fail to transfer EAE due to their inability to initiate lesions in the CNS. LPS, working through an antigen-nonspecific cell or cell products, can correct the defect in the Le-R cells such that the antigen-specific cells become capable of initiating CNS lesions which lead to development of clinical EAE.

Prostaglandin and thromboxane levels in central nervous system tissues from rats during the induction and development of experimental allergic encephalomyelitis (EAE)

Prostaglandin (PG) and thromboxane levels were measured in the spinal cords and cerebellums of rats during the induction, disease and recovery periods of experimental allergic encephalomyelitis (EAE). In spinal cords PGE and 6-oxo-PGF1α increased to maximum with the onset of neurological symptoms, 11–12 days after inoculation. However, the levels returned to normal at the height of clinical disease, despite the persistence of inflammatory lesions. After an initial fall, PGF2α increased to normal limits, 11–12 days after inoculation, and remained at this level throughout the experiment. In contrast, the cerebellum content of all the eicosanoids decreased prior to the appearance of clinical EAE. PGF2α and 6-oxo-PGF1α concentrations subsequently increased but the PGE and thromboxane levels remained depressed for the duration of the study. The role of the eicosanoids in modulating the immune response to neuroantigen is discussed together with our recent findings in guinea pigs with acute EAE.

Modulation of experimental allergic encephalomyelitis in lewis rats by monoclonal anti-T cell antibodies

The effect of monoclonal antibodies to different T lymphocyte populations of the rat on the induction and the course of experimental allergic encephalomyelitis (EAE) was investigated. EAE was induced by injection of guinea pig spinal cord in adjuvant. Subcutaneous injections of monoclonal antibodies to all peripheral T lymphocytes (W3/13) abrogated or prevented the development of clinical EAE. Similar results were obtained in animals injected with monoclonal antibodies to T helper cells (W3/25) mixed with monoclonal antibodies to T nonhelper cells (OX8). Animals treated with either W3/25 or OX8 developed clinical EAE as the control rats (subcutaneous injected with normal mouse serum). Histological examination after the acute stage revealed no significant differences between rats treated prophylactically with W3/13, W3/25 or OX8 and rats injected with normal mouse serum. Animals treated prophylactically with a mixture of W3/25 and OX8 developed, on the whole, EAE with less histological severity compared to the control animals. Treatment of rats after the onset of the first clinical symptoms of EAE (tail flaccidity) with W3/13 resulted in a less fatal course of the disease. Compared to surviving rats injected with mouse serum (controls) the number of infiltrates were reduced in these rats treated therapeutically.

Effect of treatment with copolymer 1 (Cop-1) on the in vivo and in vitro manifestations of experimental allergic encephalomyelitis (EAE)

Injections of Copolymer 1 (Cop-1), a synthetic cathodic polymer, have been reported to prevent and treat successfully acute and recurrent EAE and has been employed in patients with multiple sclerosis (MS). It has been suggested that the therapeutic effect is due to cell-mediated immune (CMI) cross-reactivity between Cop-1 and myelin basic protein (MBP), the antigen that induces EAE. We found that Cop-1 treatment of guinea pigs (GP) sensitized with MBP in adjuvant (20 μg-animal): (a) lowers the incidence of clinical disease ( 8 20 vs 14 15 ); (b) decreases severity of disease in affected GP; (c) has little effect on pathologic lesions (mean pathology index ± SEM: 1.2 ± 0.2 vs 1.6 ± 0.3; P > 0.1). Lymphocytes of MBP-sensitized GP treated with Cop-1 exhibited in vitro proliferative responses to MBP equivalent to lymphocytes of untreated EAE-GP (14,134 ± 6,532 vs 11,821 ± 3,874; mean cpm ± SEM). GP sensitized to MBP or Cop-1 (100 μg/animal) showed reactivity to the sensitizing antigen but little in vitro reactivity to the other antigen. There was no correlation between the in vitro lymphocytes response to MBP and Cop-1 in individual GP. Treatment of MBP sensitized GP with calf-thymus histone (CTH) also resulted in a lower incidence of clinical EAE with less severe disease in affected GP. There was little effect on the pathologic index and no evidence of either inhibition of MBP-induced lymphocyte proliferative responses or cross-reactivity between MBP and CTH. Thus, treatment with Cop-1 or CTH inhibits clinical manifestations of acute EAE without suppressing inflammatory cell infiltrates or sensitization to MBP.

The influence of cyclosporin A on the development of actively induced and passively transferred experimental allergic encephalomyelitis

The immunosuppressive effects of cyclosporin A (CsA) were tested on actively induced and passively transferred experimental allergic encephalomyelitis (EAE). Actively induced EAE could be inhibited if CsA was administered per os at 25 mg/kg/day but not at 10 mg/kg/day. Passive transfer of clinical EAE occurred in all cell recipients including those fed CsA at either 25 or 50 mg/kg/day. Cyclosporin A could inhibit the development of transfer active cells in vitro and in vivo, however, inhibition of transfer active populations by CsA required the presence of CsA during the initial stage of cell response. If CsA was added to Con A-stimulated spleen cell cultures after a delay of 24 hr then these cells transferred clinical disease. Similarly, animals fed CsA concurrently with basic protein sensitization did not develop cell populations capable of transferring EAE. If CsA feeding commenced 2 or 4 days following sensitization all basic proteinsensitized animals still failed to develop EAE; however these latter groups of animals were a suitable source of cells capable of transferring some signs of clinical EAE.

Unresponsiveness to experimental allergic encephalomyelitis in mice: replacement of suppressor cells by a soluble factor.

A soluble suppressor factor has been prepared from cells of mice rendered nonsusceptible to experimental allergic encephalomyelitis (EAE) by treatment with mouse spinal cord homogenate in incomplete Freund's adjuvant. The specific activity of this factor can be augmented by using a cell population enriched on plates coated with anti-mouse Fab and the specific antigen, mouse basic encephalitogen (MBE). The resultant suppressor factor had the same biologic activities as the cells from which it originated. Thus, it suppressed specifically the delayed-type hypersensitivity (DTH) response to MBE in vivo, and blocked in vitro the effector lymphocytes that adoptively transfer the DTH response. The suppressor factor reactivity was manifested also by the capacity to suppress the activity of macrophage migration inhibition factor produced by sensitized lymphocytes in the presence of the specific antigen MBE. The suppressor factor is antigen-specific and can bind the MBE in vitro and thus compete with its antibody binding. The most significant activity of the soluble suppressor factor is its ability to interfere with the induction of clinical EAE.

Experimental allergic encephalomyelitis induced by basic protein with synthetic adjuvant in comparison with Freund's complete adjuvant: Role of antibodies in correlation with the clinicopathological features

Experimental allergic encephalomyelitis (EAE) was induced in guinea pigs with bovine myelin basic protein (BP) with adjuvant of either synthetic muramyl dipeptide (Mdp) or Mycobacterium tuberculosis (Tbc). The following results were obtained: (1) The body temperature of the animals was studied serially after sensitization and its elevation was shown to be an early sign of EAE. (2) Several animals developed the clinical and histological signs of hyperacute EAE. (3) An optimal combined dosage of BP and adjuvant was found for induction of clinical EAE and for the production of complement fixing (CF) antibodies. (4) Little passive hemagglutinating (PH) antibody was produced by single immunization. These results displayed no essential difference in EAE induced by either adjuvant. (5) Detectable PH antibodies developed later in addition to CF antibodies in a few animals immunized with Tbc adjuvant. These animals were skin-tested to BP, and had recovered from body weight loss or limb weakness. The results suggest that humoral antibodies play a role in modifying the disease process, even if they are not essential in production of EAE.

Liposomes containing myelin basic protein (BP) suppress but do not induce allergic encephalomyelitis in Lewis rats.

The ability of rats to respond immunologically to myelin basic protein (BP) incorporated into liposomes was tested. BP-liposomes administered intradermally or intravenously to naive rats were non-encephalitogenic, did not induce antibody or cellular reactivity to BP, nor did they protect against a subsequent challenge with BP in complete Freund's adjuvant (CFA). Intravenous administration of BP-liposomes to sensitized rats, however, suppressed EAE if given shortly before the expected onset of clinical signs. The same dose of BP in saline fails to suppress the appearance of clinical experimental allergic encephalomyelitis (EAE).

Suppression of experimental allergic encephalomyelitis with thoracic duct lymphocytes.

Thoracic duct lymphocytes (TDL) from Lewis rats immunized 9-10 days previously with basic protein in complete Freund's adjuvant (BP-CFA) failed to induce experimental allergic encephalomyelitis (EAE) in syngeneic recipients. This contrasts with the successful transfer of EAE by lymph node cell suspensions from donors immunized 9 days previously with BP-CFA. Only minor EAE was induced passively by TDL from rats immunized 11-12 days before with BP-CFA. TDL collected 9-20 days after BP-CFA immunization, however, were successful in transferring specific suppression of EAE tested by the lack of disease in the recipients immunized actively with BP-CFA 1 week after the TDL transfer. The data indicate that the thoracic duct contains specific suppressor cells shortly before, during and after the development of clinical EAE.

Suppression of Experimental Allergic Encephalomyelitis by 6-Hydroxyphthalaldehydic Acid, O-(p-Chlorobenzyl)Oxime (EN3638)

Abstract EN3638 is a new oxime derivative of salicylic acid that has immunosuppressive properties. Oral administration of the compound to rats during the incubation period of experimental allergic encephalomyelitis (EAE) delayed the onset of clinical signs. EN3638 was effective in both ordinary and hyperacute forms of EAE. Doses of 50, 100, or 150 mg/kg daily, 250 mg/kg three times a week, or 400 mg/kg twice a week suppressed both clinical and histologic evidences of EAE during the course of therapy (as long as 4 weeks) and for 8 or more days thereafter. Clinical EAE developed after a full incubation period after discontinuance of EN3638, probably due to the persisting depot of antigen in oil. When EAE was produced without an oily depot, a single dose suppressed the disease for at least 5 weeks in some rats. EN3638 was effective when given only in the second half of the incubation period but not when given at the time that EAE lesions and signs develop. Passive transfer experiments suggested that the drug prevented and even reversed sensitization to neural antigens. It had only slight effect on fully sensitized lymphoid cells or on the recruitment of nonimmune inflammatory cells in the nervous system, and it was not acting as a source of salicylate or as an adrenocortical stimulator.

Immunoregulation of Experimental Allergic Encephalomyelitis: Conditions for Induction of Suppressor Cells and Analysis of Mechanism

We determined requirements for the induction of immunoregulatory suppressor cells in experimental allergic encephalomyelitis (EAE) in Lewis rats. Pretreatment of rats with myelin basic protein (BP) in incomplete Freund's adjuvant (IFA) stimulates the proliferation of suppressor cells that localize in lymph nodes and spleen (but not thymus) and exert control over the development of clinical EAE. Dosage studies revealed that 3 X 10(7) suppressor cells can adoptively transfer suppression to syngeneic recipients. Transferred unresponsiveness wanes within 3 weeks, indicating that the suppressor cells are short-lived lymphocytes, although actively induced unresponsiveness persists for at least 8 weeks, probably as a result of continual proliferation under the influence of antigen. No evidence was obtained to suggest that antigen carry-over or blocking antibody production accounts for adoptive transfer of unresponsiveness. Suppressor cells apparently act at the inductive phase of the immune response since they had no inhibitory effect on adoptive transfer of disease by effector lymph node cells. Other mechanisms also may play a role in unresponsiveness to EAE, since rats pretreated i.v. with high dosages of soluble BP were temporarily rendered unresponsive, although suppressor cells could not be detected in these animals.

Protection Against Experimental Allergic Encephalomyelitis in Guinea Pigs by Bacterial Lipopolysaccharide

Protection against experimental allergic encephalomyelitis (EAE) was studied in guinea pigs. Antibody titers to bovine myelin basic protein (BP), measured by means of passive hemagglutination on day 12 or 13 after sensitization with bovine spinal cord (BSC) in complete Freund’s adjuvant (CFA), were higher in animals which recovered later from mild EAE than in those which showed severe EAE. The effect of bacterial lipopolysaccharide (LPS), a B lymphocyte stimulator, on the suppression of EAE was examined. Pretreatment of guinea pigs with LPS 3 days prior to the sensitization with BSC-CFA at the same sites resulted in marked attenuation of both clinical and histologic EAE. The protective effect of LPS against EAE, however, depended upon the times and sites of its use; LPS suppressed EAE only when it was given 3 days prior to the sensitization with BSC-CFA at the same sites. Intraperitoneal injection of LPS, even if given 3 days prior to sensitization, failed to suppress EAE. In the group of guinea pigs protected against EAE by pretreatment with LPS, delayed hypersensitivity skin reactions to BP were depressed and humoral antibody titers to BP were increased several days earlier than in the control group although their peak titers were not significantly different. The ratio of T to B lymphocytes, as assessed by rosette formation, was diminished in the draining lymph nodes of guinea pigs protected against EAE, indicating an increased proliferation of B lymphocytes. These results suggested that the stimulation and proliferation of B lymphocytes themselves or their products, antibodies to BP, could inhibit T lymphocytes and suppress EAE.

Characterization of suppressor cells involved in regulation of experimental allergic encephalomyelitis

AbstractThis report describes studies carried out in an effort to identify the suppressor cells involved in the regulation of experimental allergic encephalomyelitis (EAE) in the Lewis rat. Viable lymph node cells (LNC) from donors rendered tolerant to clinical EAE by pretreatment with myelin basic protein antigen in nonencephalitogenic form are capable of transferring unresponsiveness to syngeneic recipients. Adoptive transfer of unresponsiveness was abrogated if LNC were treated in vitro with anti‐thymocyte serum and complement.In further experiments, it was shown that suppressor cells are among the LNC subpopulations that do not adhere to glass wool. These findings suggest that the immunoregulatory cells in EAE are suppressor T lymphocytes.

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 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.

Antigen-induced inhibition of experimental allergic encephalomyelitis. IV. Studies of the C-terminal end of the myelin basic protein molecule (1).

The C-terminal end of the myelin basic protein (BP) molecule (peptide 117.170) was obtained by cleavage with BNPS-skatole, and purified by gel filtration. Peptide 117-170 from guinea pig BP induced experimental allergic encephalomyelitis (EAE) in Lewis rats, whereas the corresponding peptide from bovine BP was inactive. Neither peptide showed more than trace activity when tested in guinea pigs. Guinea pigs and rats treated with peptide 117-170 in incomplete Freund adjuvant were not rendered unresponsive to EAE induced by subsequent challenge with an encephalitogenic emulsion of BP in complete Freund adjuvant. Indeed, peptide-pretreated rats tended to develop clinical EAE significantly earlier than untreated controls.