Evaluation of a radiolabelled peripheral benzodiazepine receptor ligand in the central nervous system inflammation of experimental autoimmune encephalomyelitis: a possible probe for imaging multiple sclerosis.

PURPOSE: Peripheral benzodiazepine receptors (PBRs) are upregulated on macrophages and activated microglia, and radioligands for the PBRs can be used to detect in vivo neuroinflammatory changes in a variety of neurological insults, including multiple sclerosis. Substituted 2-phenyl imidazopyridine-3-acetamides with high affinity and selectivity for PBRs have been prepared that are suitable for radiolabelling with a number of positron emission tomography and single-photon emission computed tomography (SPECT) isotopes. In this investigation, the newly developed high-affinity PBR ligand 6-chloro-2-(4'-iodophenyl)-3-(N,N-diethyl)imidazo[1,2-a]pyridine-3-acetamide, or CLINDE, was radiolabelled with 123I and its biodistribution in the central nervous system (CNS) of rats with experimental autoimmune encephalomyelitis (EAE) evaluated. METHODS: EAE was induced in male Lewis rats by injection of an emulsion of myelin basic protein and incomplete Freund's adjuvant containing Mycobacterium butyricum. Biodistribution studies with 123I-CLINDE were undertaken on EAE rats exhibiting different clinical disease severity and compared with results in controls. Disease severity was confirmed by histopathology in the spinal cord of rats. The relationship between inflammatory lesions and PBR ligand binding was investigated using ex vivo autoradiography and immunohistochemistry on rats with various clinical scores. RESULTS: 123I-CLINDE uptake was enhanced in the CNS of all rats exhibiting EAE when compared to controls. Binding reflected the ascending nature of EAE inflammation, with lumbar/sacral cord>thoracic cord>cervical cord>medulla. The amount of ligand binding also reflected the clinical severity of disease. Ex vivo autoradiography and immunohistochemistry revealed a good spatial correspondence between radioligand signal and foci of inflammation and in particular ED-1+ cells representing macrophages and microglia. CONCLUSION: These results demonstrate the ability of 123I-CLINDE to measure in vivo inflammatory events represented by increased density of PBRs and suggest that 123I-CLINDE warrants further investigation as a potential SPECT marker for imaging of CNS inflammation.

Inhibition of nitric oxide synthase initiates relapsing remitting experimental autoimmune encephalomyelitis in rats, yet nitric oxide appears to be essential for clinical expression of disease.

Myelin basic protein-CFA-induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats is an acute monophasic disease from which animals recover. In this model, spontaneous relapses do not occur and rats develop a resistance to further active reinduction of disease. Previously, we reported that oral administration of the NO synthase inhibitor N-methyl-L-arginine acetate (L-NMA) to recovered rats precipitated a second episode of disease in 100% of animals. Further studies now show that this second clinical episode is actually a chronic relapsing disease that persists for months. This occurs only in rats that have recovered from actively induced EAE and not in rats recovered from passively induced EAE, suggesting the need for a peripheral Ag depot to induce secondary disease. We have also determined that clinical signs of EAE in L-NMA-treated recovered rats do not appear until L-NMA treatment has stopped. This is despite the fact that, at the same time point, CNS inflammatory lesions in symptomless animals receiving L-NMA are qualitatively and quantitatively similar to those with severe disease symptoms from whom L-NMA treatment has been withdrawn. The latter animals have significantly higher levels of reactive nitrogen intermediates in the cerebrospinal fluid than the former group. This study examines the mechanism of reinduction of disease by L-NMA treatment, and the findings suggest a dual role for NO in regulation of pathology in EAE that is dependent on site and timing of NO production.

Cell surface expression of the 300 kDa mannose-6-phosphate receptor by activated T lymphocytes.

Phosphosugars, such as mannose-6-phosphate (M6P), have been shown previously to display anti-inflammatory properties, notably inhibition of experimental autoimmune encephalomyelitis (EAE) and adjuvant-induced arthritis in rats. It has been proposed that M6P exerts its anti-inflammatory effect by displacing lysosomal enzymes, which are involved in T-cell extravasation into inflammatory sites, from the 300 kDa mannose-6- phosphate receptor (MPR-300) on the surface of T cells. If this model is correct MPR-300 should be selectively expressed on the surface of activated T cells, as T cell entry into the central nervous system in EAE depends on the T cells being in an activated state. Thus, the present study examines whether cell surface expression of MPR-300 by T lymphocytes correlates with their state of activation and whether T cells in inflammatory sites express the receptor. Flow cytometric studies showed MPR-300 to be absent from the surface of unstimulated rat T cells isolated from peripheral blood and lymphoid tissues, and T cells resident within the peritoneal cavity. In contrast, MPR-300 was expressed on activated T cells derived from an inflammatory peritoneal exudate. In vitro studies demonstrated transient expression of MPR-300 on the surface of splenic T cells following stimulation with Con A. MPR-300 was also induced on T-cell lines by antigen stimulation. These data demonstrate that T cells in inflammatory sites express MPR-300 on their surface and activation of T lymphocytes induces cell surface expression of MPR-300. Such findings are consistent with the hypothesis that cell surface MPR-300 is required for the entry of T cells into inflammatory sites.

Nitric oxide plays a critical role in the recovery of Lewis rats from experimental autoimmune encephalomyelitis and the maintenance of resistance to reinduction.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease of the CNS and an animal model for the human demyelinating disease, multiple sclerosis. In the Lewis rat, myelin basic protein (MBP)-CFA-induced EAE is an acute monophasic disease from which animals recover fully, do not relapse, and develop a robust long-term resistance to further active reinduction of disease. In this paper, we report that rats recovering from MBP-CFA-induced EAE have significantly increased serum levels of reactive nitrogen intermediates indicative of increased NO production. These levels remain elevated after the recovery period and increase even further early after a rechallenge with MBP-CFA, and all animals are totally refractory to a second episode of disease. Oral treatment of rats with N-methyl-l -arginine acetate (l -NMA), beginning at peak disease on day 11 postimmunization, results in significant prolongation of disease and an alteration in the presentation of clinical symptoms from that of solely hind limb paresis/paralysis to severe fore limb involvement as well. Treatment of fully recovered rats with l -NMA 24 h before a rechallenge with MBP-CFA leads to decreased serum reactive nitrogen intermediate levels and results in a second episode of EAE in 100% of animals. Furthermore, l -NMA treatment of fully recovered rats in the absence of a rechallenge immunization leads to spontaneous relapse of disease.

IFN-gamma is critical to the control of murine autoimmune encephalomyelitis and regulates both in the periphery and in the target tissue: a possible role for nitric oxide.

NO and IFN-gamma have normally been considered cytotoxic and proinflammatory molecules, respectively, in the setting of the central nervous system inflammatory disease autoimmune encephalomyelitis (EAE). Using mice lacking the ligand binding chain of the IFN-gamma receptor (IFNgammaR-/-), we have previously shown that IFN-gamma is not essential for myelin oligodendrocyte glycoprotein peptide (MOG35-55) induced EAE expression but is in fact essential for its down-regulation. Here we examined the downstream molecular and cellular mechanism(s) of IFN-gamma regulation and demonstrate that neither IL-4 nor IL-10 appear to play a role in down-regulation nor do various lymphoid cell populations. Cells of the macrophage lineage are key to down-regulation as evidenced by the fact that peritoneal exudate cells from IFNgammaR+/+ mice inhibit Ag-driven proliferation of IFNgammaR-/- lymphocytes, whereas IFNgammaR-/- peritoneal exudate cells do not. High levels of reactive nitrogen intermediates are detected in the former cultures but not the latter, and the inhibition of proliferation is reversible with an inhibitor of inducible NO synthase, indicating a key role for NO in down-regulation. Studies with bone marrow chimeras indicate that down-regulation occurs not only systemically but also within the target tissue. These data suggest that IFN-gamma down-regulates EAE by inducing inducible NO synthase and subsequently NO production, both by macrophages in the periphery and, by inference, microglia and astrocytes in the target tissue.

Our shifting understanding of the role of nitric oxide in autoimmune encephalomyelitis: a review.

Nitric oxide was first described being produced in inflammatory cells involved in experimental autoimmune encephalomyelitis in 1992. Since then some 45 papers have appeared examining the role of NO in this central nervous system autoimmune inflammatory disease. Of the first 10 papers published all resulted in the interpretation that NO was a pathologic or "bad" molecule in the context of EAE. A few papers then began to appear suggesting that NO may not in fact always be a harmful molecule and by the end of 1997 early 1998, 22 papers suggested a destructive role for the molecule while three suggested it was protective. The past two years have seen a significant increase in reports supporting a protective mechanism for NO in EAE such that as of July 1999, 27 papers suggest a destructive and 15 a protective role for NO with a few uncommitted. This review sets out in a more or less chronological order the studies examining the role of NO in EAE and maps our changing understanding of the molecules role in this CNS inflammatory disease and by inference perhaps multiple sclerosis.

Treatment with anti-granulocyte antibodies inhibits the effector phase of experimental autoimmune encephalomyelitis.

Emerging data suggest that polymorphonuclear leukocytes (PMNLs) can play an important role in Ag-dependent immune responses. Therefore, we have assessed the involvement of these cells in the development of an organ-specific autoimmune disease, experimental autoimmune encephalomyelitis (EAE), in the mouse. Depletion of peripheral blood PMNLs beginning day 8 after immunization significantly delayed and in some cases totally prevented the development of clinical EAE in mice. Depletion of PMNLs beginning 1 day before sensitization and continuing until day 7 postimmunization had no effect on the subsequent development of EAE, suggesting that depletion alters the efferent but not the afferent arm of the immune response. In vitro studies showed that lymphoid cells from mice protected from EAE by PMNL depletion beginning on day 8 postsensitization proliferated in response to specific Ag to a level equal to cells from sensitized animals treated with control serum, again indicating that treatment was not affecting the afferent limb of the immune response. Further evidence that PMNL may be necessary in initiating the pathology of EAE was seen in passive transfer experiments where PMNL-depleted recipients of MBP-specific lymphoid effector cells developed EAE much less effectively than did animals treated with control Ab. Taken together, these data indicate that PMNLs play a critical role in the effector phase of the development of the clinicopathologic expression of EAE in mice.

Nitric oxide is a potential down-regulating molecule in autoimmune disease: inhibition of nitric oxide production renders PVG rats highly susceptible to EAE.

Rat strains vary in their susceptibility to experimental autoimmune encephalomyelitis (EAE) and in many cases, factors other than MHC antigens are thought to play a role in this. We found that PVG rats, which have a very low susceptibility to EAE, were rendered highly susceptible to clinical disease when treated with N-methylarginine (NMA) an inhibitor of nitric oxide synthase (NOS). The clinical course of the ensuing disease in NMA-treated PVG rats was in most cases fulminating in nature and accompanied by some mortality. Following immunisation with myelin basic protein (MBP)-complete Freund's adjuvant (CFA), PVG rats developed higher serum levels of the surrogate markers of nitric oxide production, reactive nitrogen intermediates (RNI; nitrite and nitrate), than did their Lewis counterparts. This in vivo finding was reflected in vitro, where the levels of RNI produced in 24, 48 and 72 h IFN-gamma-stimulated spleen cell cultures for PVG rats were significantly higher than those for Lewis rats. A mechanism by which increased NO production might protect PVG rats against clinical EAE was suggested by the finding that lymph node cells, isolated from NMA-treated MBP-immunised PVG rats, proliferated in response to MBP at a rate approximately 3 x greater than those from MBP-immunised, saline treated rats. Thus, the greater number of MBP-specific T cells generated in the NOS inhibitor-treated vs. untreated rats could account for their increased susceptibility to developing clinical EAE. The findings in this study suggest that NO plays a role in protecting PVG rats against developing EAE.

Approaches to the treatment of central nervous system autoimmune disease using specific neuroantigen.

The ultimate aim in the treatment of autoimmune disease is to restore self-tolerance to the autoantigen(s) in question. In lieu of this ideal result, the conversion of a destructive or pathogenic autoimmune response into one of benign autoimmunity would also be highly desirable. In either case the use of the antigenic epitope, which is the target of the destructive immune response, would ideally be employed so as to give specificity to the protection without the need for long-term immunosuppression. This review describes a number of different approaches using various forms, doses, and routes of injection of specific neuroantigen to inhibit the different clinical varieties of autoimmune encephalomyelitis in a number of animal models; all done with the view to translating the findings into the clinic for the treatment of multiple sclerosis. We conclude that any treatment strategy for multiple sclerosis (MS) must have a number of features: it must be clinically acceptable, specific, long-lasting, require only short-term treatment, able to shunt off ongoing disease, and have the potential to prevent or deal with epitope spreading. Few of the approaches we describe fulfill all of these criteria. We suggest that investigations of new adjunctive agents to be used with a specific antigen be pursued, and that currently the use of chimeric proteins or DNA vaccination with or without the new adjunctives may hold the most hope for the future.

Treatment of central nervous system inflammation with inhibitors of basement membrane degradation.

Currently available anti-inflammatory drugs for the treatment of multiple sclerosis (MS) and other inflammatory diseases are generally inadequate, with disease progression not being arrested by the treatments and undesirable side effects posing problems. In response to these deficiencies our laboratories have, over the past 10 years, been developing novel drugs that interfere with the entry of leucocytes into inflammatory sites by inhibiting their passage through the subendothelial basement membrane (BM). This review initially summarizes evidence supporting the hypothesis that the subendothelial BM is a major barrier to the accumulation of leucocytes in inflammatory sites. An important point that has emerged is that breaching of the BM is probably a cooperative process, involving activation- and cytokine-induced degradative enzymes contributed by leucocytes, endothelial cells and platelets. The review then discusses the properties of three separate classes of anti-inflammatory compounds we have developed, namely sulfated polysaccharides/oligosaccharides, phosphosugars, and castanospermine (CS), which inhibit the passage of leukocytes through BM. Each drug type appears to prevent BM degradation by a different mechanism. Sulfated polysaccharides/oligosaccharides mediate their anti-inflammatory effect by inhibiting the endoglycosidase, heparanase, which plays a key role in the solubilization of BM by invading leucocytes. In fact, our studies have highlighted the heparanase enzyme as a major target for future drug development. Phosphosugars probably inhibit inflammation by displacing lysosomal enzymes, which are involved in BM degradation, from cell surface mannose 6-phosphate receptors. This mechanism of expressing degradative enzymes on the cell surface is particularly evident with activated T lymphocytes. On the other hand, CS interferes with appropriate targeting of lysosomal enzymes involved in BM degradation. For reasons which are still unclear, CS specifically inhibits BM degradation by endothelial cells, which results in a characteristic perivascular arrest of leucocytes in inflammatory sites. Overall, our studies have established that inhibitors of subendothelial BM degradation represent viable anti-inflammatory agents. It is hoped that future work will result in the development of a totally new class of highly effective, subtle and non-toxic anti-inflammatory drugs for the treatment of MS and other inflammatory diseases.

DNA vaccines for the treatment of autoimmune disease.

DNA vaccines represent one of the most significant developments in vaccine technology in recent years. Although, in general, studies have primarily focused on the induction of protective immune responses against infectious pathogens, the technology may prove useful for other immune-related diseases, including autoimmunity. Autoimmune disease results from a breakdown in tolerance to self antigens; however, the same fundamental immunological reactions that control immune responses to foreign antigens are also likely to operate during the course of autoimmune disease. These include the reciprocal regulation of Th cell subsets. Th1 cells appear to be involved in many organ-specific autoimmune diseases while suppression of disease is associated with cells of the Th2 phenotype. It has been possible, therefore, to suppress many of the pathological consequences of autoimmunity by manipulating the Th1/Th2 cell balance. The induction of Th2 responses by DNA immunization might therefore be expected to have a profound effect on the course of autoimmune disease. Indeed, we have demonstrated that DNA immunization can protect animals against the autoimmune central nervous system inflammatory disease, experimental autoimmune encephalomyelitis (EAE). As many other autoantigens have now been identified, the application of this technology to other autoimmune diseases warrants investigation.

Infusion of soluble myelin basic protein protects long-term against induction of experimental autoimmune encephalomyelitis.

Protection against experimental autoimmune encephalomyelitis (EAE) induced by s.c. infusion of myelin basic protein (MBP) alone is dose dependent and long lived. Protection is not effective against passively induced disease nor is it transferable with lymphoid cells. The proliferative response of lymph node cells to MBP following encephalitogenic challenge is decreased in the EAE-protected animals as is the production of IL-2 and IFN-gamma by these cells. Treatment with soluble MBP promed rats for antibody production is evidenced by the early appearance of anti-MBP antibody following encephalitogenic challenge. Determination of antibody isotype following challenge revealed a change in the ratio of IgG1 to IgG2a with a significant increase in the amount of IgG1 produced. These data suggest that infusion of high dose soluble neuroantigen primes the immune response such that subsequent challenge with an encephalitogenic inoculum pushes the response down a non-destructive Th2 autoimmune pathway.

Castanospermine, an oligosaccharide processing inhibitor, reduces membrane expression of adhesion molecules and prolongs heart allograft survival in rats.

The inhibition of intracellular oligosaccharide processing is a new approach to immunosuppression in allotransplantation. The net effect of such inhibition is reduction in the membrane expression of certain glycoproteins. Hence cell-cell interaction in allorejection may be impaired in the presence of glycoprotein processing inhibitors because the expression of key ligand-receptor pairs of N-linked glycoproteins including adhesion molecules is inhibited. The aims of this study were to measure the immunosuppressive ability of castanospermine (CAST) in a rat heart allograft model, to measure its effect on membrane expression of adhesion molecules (LFA-1 alpha, LFA-1 beta, ICAM-1), class I and class II MHC antigens and on other T cell associated molecules (CD4, CD8, CD39, CD45, W3/13), to test its tolerogenic potential and its toxicity. Membrane expression of these molecules was measured by flow cytometry for single cells and by immunoperoxidase staining for the allograft. In grafted rats CAST significantly reduced the expression of LFA-1 alpha on lymphoid cells in the thymus, lymph node, spleen and heart allografts. ICAM-1 expression on endothelial cells of the allograft vasculature, class I and class II MHC expression on lymphoid cells in the thymus, class II MHC expression on lymphoid cells in the allograft; and CD4, CD8, CD45 and W3/13 expression on lymphoid cells in some organs. By contrast, in non-grafted rats CAST significantly upregulated expression of class I MHC and CD45 in the thymus, lymph node and spleen, ICAM-1 and CD4 on lymphoid cells in the spleen, but reduced expression of LFA-1 alpha on lymphoid cells in the thymus. It also prolonged rat heart allograft survival in a dose-dependent manner and with limited testing was relatively non-toxic. In conclusion, CAST is an immunosuppressive molecule which may work by downregulation of the ligand-receptor adhesion molecule pair, LFA-1 alpha-ICAM-1 although subtle downregulation of class I and II MHC, CD4 and CD8 molecules could also contribute to its immunosuppressive activity. Hence, both lymphocyte-endothelial cell binding and lymphocyte activation may be inhibited by CAST. This work suggests that CAST may hold significant potential as a transplant immunosuppressant probably as an adjuvant agent to inhibitors of interleukin 2 secretion.

IFN-gamma plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis.

129/Sv mice are resistant to induction of experimental autoimmune encephalomyelitis (EAE) induced with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Mice of this strain lacking the gene coding for the ligand-binding chain of the IFN-gamma receptor develop EAE with high morbidity and mortality. Spleen cells from sensitized IFN-gammaR-/- mice proliferated extensively when stimulated with MOG peptide in culture and produced high levels of IFN-gamma and TNF but no detectable IL-4. Transfer of spleen cells from sensitized IFN-gammaR-/- mice produced EAE in both IFN-gammaR+/+ and IFN-gammaR-/- recipients. Disease was severe in IFN-gammaR-/- recipients and mortality high (77%). Surviving mice remained moribund until termination of the experiments. IFN-gammaR+/+ recipients developed disease of equal severity, but with no mortality, and recovered significantly. These results indicate that IFN-gamma is not essential for the generation or function of anti-MOG35-55 effector cells but does play an important role in down-regulating EAE at both the effector and induction phase of disease.

Short term treatment with soluble neuroantigen and anti-CD11a (LFA-1) protects rats against autoimmune encephalomyelitis: treatment abrogates autoimmune disease but not autoimmunity.

Resistance to autoimmune encephalomyelitis was induced by s.c. infusion of myelin basic protein (MBP) in saline in combination with i.p. injections of anti-CD11a (LFA-1) mAbs. This treatment induces resistance to EAE induction, which appears early and persists for at least one month after treatment. Some MBP-CFA-challenged resistant rats showed minimal inflammation in the central nervous system, which was, however, confined to the meninges of the lower spinal cord. Examination of the immune status of MBP-anti-LFA-1 treated rats before encephalitogenic challenge failed to reveal any priming when assessed by Ag driven proliferation and cytokine production by lymphoid cells, and by circulating Ab production. Following challenge of protected rats, lymph node cell proliferation to MBP was unaltered, indicating that reactive cells had not been deleted or energized. Resistance could not be transferred with lymphoid cells from treated rats nor abrogated by cyclophosphamide treatment. In treated rats following challenge, there was a shift in the isotype of anti-MBP Ab produced, from an IgG2a:IgG1 ratio of 2:1 to 1:1, due to an increase in IgG1 production, indicating a possible bias towards a nonpathogenic Th2 CD4+ T cell response. The IgG1 Ab was detected early after challenge suggesting that pretreatment had indeed primed the animals, and had primed them to go down the Th2 pathway following encephalitogenic challenge. The ability to divert immune reactivity from a destructive to a nondestructive response could have important therapeutic implications for autoimmune disease.

Suppression of hyperacute and passively transferred experimental autoimmune encephalomyelitis by the anti-oxidant, butylated hydroxyanisole.

Butylated hydroxyanisole (BHA) was used to treat hyperacute, ordinary passive, and hyperacute passive experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. The anti-oxidant, delivered via mini-osmotic pumps, reduced the incidence, severity and mortality in hyperacute EAE and also reduced the incidence, severity and duration of disease in passively induced EAE and hyperacute passive EAE. In all cases, cellular infiltration by both mononuclear and polymorphonuclear leukocytes were significantly reduced in treated rats. BHA appears therefore to act at the effector stage of EAE, reducing cellular infiltration in the brain and spinal cord and minimising clinical signs without blocking sensitisation or activation. This was supported by the finding that spleen cells from BHA-treated donors immunised for hyperacute EAE transferred disease at least as well as cells recovered from untreated donors.

Studies on T-cell receptors involved in experimental autoimmune encephalomyelitis using the complementary peptide recognition approach.

Based upon Blalock's complementary recognition approach, a complementary or antisense peptide (CP) was designed to the experimental autoimmune encephalomyelitis (EAE) epitope peptide, rat myelin basic protein (MBP) peptide 72-82. This peptide (EAE CP) was shown to have some sequence similarities to T-cell receptors (TCR) and MHC II molecules in a sequence homology search. Solid-phase binding assays demonstrated specific and high affinity binding (3 and 4 microM) between the EAE CP and the rat and guinea pig EAE epitope peptides (Rt72-82 and Gp69-82), respectively. This EAE CP was also found to be immunogenic in rats in an ear swelling test for delayed type hypersensitivity (DTH) reactions and an ELISA for antibody responses. However, a rabbit antibody generated to EAE CP was shown to be unable to stain the V beta 8+ EAE susceptible T-cells in immunofluorescence analyses. This EAE CP was also used in attempts to down-regulate EAE and the results showed that prior immunization with EAE CP in complete Freund's adjuvant could not prevent the Lewis rats from developing EAE. Although the data on sense-antisense peptide interaction were positive and the EAE CP was immunogenic, the inability of EAE CP to regulate EAE indicates that the CP approach may not be generally applicable.