John L. Trotter

Aminoguanidine, an inhibitor of inducible nitric oxide synthase, ameliorates experimental autoimmune encephalomyelitis in SJL mice.

Previous work from our laboratory localized nitric oxide to the affected spinal cords of mice with experimental autoimmune encephalomyelitis, a prime model for the human disease multiple sclerosis. The present study shows that activated lymphocytes sensitized to the central nervous system encephalitogen, myelin basic protein, can induce nitric oxide production by a murine macrophage cell line. Induction was inhibited by amino-guanidine, a preferential inhibitor of the inducible nitric oxide synthase isoform, and by NG-monomethyl-L-arginine. Aminoguanidine, when administered to mice sensitized to develop experimental autoimmune encephalomyelitis, inhibited disease expression in a dose-related manner. At 400 mg aminoguanidine/kg per day, disease onset was delayed and the mean maximum clinical score was 0.9 +/- 1.2 in aminoguanidine versus 3.9 +/- 0.9 in placebo-treated mice. Histologic scoring of the spinal cords for inflammation, demyelination, and axonal necrosis revealed significantly less pathology in the aminoguanidine-treated group. The present study implicates excessive nitric oxide production in the pathogenesis of murine inflammatory central nervous system demyelination, and perhaps in the human disease multiple sclerosis.

B cells and antibodies in CNS demyelinating disease

There is much evidence to implicate B cells, plasma cells, and their products in the pathogenesis of MS. Despite unequivocal evidence that the animal model for MS, EAE, is initiated by myelin-specific T cells, there is accumulating evidence of a role for B cells, plasma cells, and their products in EAE pathogenesis. The role(s) played by B cells, plasma cells, and antibodies in CNS inflammatory demyelinating diseases are likely to be multifactorial and complex, involving distinct and perhaps opposing roles for B cells versus antibody.

Polyneuropathy syndromes associated with serum antibodies to sulfatide and myelin-associated glycoprotein.

We studied a series of 64 patients with sensory ± motor peripheral neuropathies by comparing clinical and physiologic features to serum antibody reactivity against compounds containing sulfated carbohydrate moieties. We determined antibody reactivity by an enzyme-linked immunosorbent assay (ELISA) using purified glycolipids and glycoproteins as antigens, and we used high-performance thin-layer chromatography and Western blotting to test the specificity of results. Twelve patients with high titers of IgM antibodies directed against the myelin-associated glycoprotein (MAG) had sensory-motor polyneuropa-thies with physiologic evidence of demyelination. IgM antibody reactivity to MAG was associated with an IgM serum M protein in five patients. Eight other patients, most with sensory >motor polyneuropathies, had high titers of antibody reactivity to sulfatide but not of IgM to MAG. TWO had an associated IgM paraprotein. None of the patients with selective serum antisulfatide activity had predominantly demyelinating features on physiologic testing. We conclude that (1) high ELISA titers of antibodies to MAG may be more common than previously suspected in patients with chronic demyelinating sensory-motor neuropathies, and (2) the presence of high titers of antisulfatide antibodies in serum may provide clues to the pathogenesis of otherwise idiopathic, axonal, predominantly sensory neuropathies.

Mediation of inflammation by encephalitogenic cells: interferon γ induction of nitric oxide synthase and cyclooxygenase 2

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated inflammatory demyelinating disorder of the central nervous system (CNS) which serves as a prime animal model for the human disease multiple sclerosis. Previous studies from these laboratories demonstrated excess nitric oxide (NO) in the CNS of EAE-affected mice, and amelioration of EAE with a selective inhibitor of the inducible nitric oxide synthase (iNOS). Recent studies from other laboratories have indicated that prostaglandin PGE2 is increased in CNS tissues of EAE-affected rodents and that EAE is prevented by the inhibition of cyclooxygenase activity. The present study investigated the ability of encephalitogenic lymphoid cells to induce NOS and cyclooxygenase (COX-2) in the murine macrophage line, RAW 264.7. In order to mimic the extracellular milieu present in EAE lesions, conditioned medium (CM) of activated EAE-inducer cells was added to this macrophage line. CM caused a time-dependent increase in nitrite, indicating NO production. Reverse-transcriptase PCR demonstrated iNOS mRNA in RAW 264.7 cells, first detected at 3 h, and Western blots confirmed the induction in RAW cells of the 130-kDa iNOS protein. Production of nitrite by CM-exposed RAW 264.7 cells was blocked by inhibitors of NOS (L-N-methylarginine or aminoguanidine) or by antibodies to murine IFN-gamma or IL-1 beta. CM of activated encephalitogenic cells induced production of PGE2 by RAW 264.7 cells, as determined by ELISA, and Western blots identified the presence of the 70-80-kDa inducible COX (COX-2) protein. Induction of COX-2 could be inhibited by antibody to IFN-gamma. Thus, encephalitogenic cells are capable of inducing the expression of the inflammatory enzymes iNOS and COX-2 in a murine macrophage line via the T cell cytokine IFN-gamma, alone or in combination with IL-1 beta.

Class II-restricted T cell responses in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease. III. Failure of neuroantigen-specific immune tolerance to affect the clinical course of demyelination

Abstract Intracerebral inoculation of Theilers murine encephalomyelitis virus (TMEV) into susceptible mouse strains produces a chronic demyelinating disease in which mononuclear cell-rich infiltrates in the central nervous system (CNS) are prominent. Current evidence strongly supports an immune-mediated basis for myelin breakdown, with an effector role proposed for TMEV-specific, major histocompatibility complex (MHC) class II-restricted delayed-type hypersensitivity (DTH) responses in which lymphokine-activated macrophages mediate bystander demyelination. The present study examined the possibility that concomitant or later-appearing neuroantigen-specific autoimmune T cell responses, such as those demonstrated in chronic-relapsing experimental allergic encephalomyelitis (R-EAE), may contribute to the demyelinating process following TMEV infection. T cell responses against intact, purified major myelin proteins (myelin basic protein (MBP) and proteolipid protein (PLP), and against altered myelin constituents were readily demonstrable in SJL/J mice with R-EAE, but were not detectable in SJL/J mice with TMEV-induced demyelinating disease. TMEV-infected mice also did not display T cell responses against the peptide fragments of MBP(91–104) and PLP(139–151) recently shown to be encephalitogenic in SJL/J mice. In addition, induction of neuroantigen-specific tolerance to a heterogeneous mixture of CNS antigens, via the i.v. injection of syngeneic SJL/J splenocytes covalently coupled with mouse spinal cord homogenate, resulted in significant suppression of clinical and histologic signs of R-EAE and the accompanying MBP- and PLP-specific DTH responses. In contrast, neuroantigen-specific tolerance failed to alter the development of clinical and histologic signs of TMEV-induced demyelinating disease or the accompanying virus-specific DTH and humoral immune responses. These findings demonstrate that TMEV-induced demyelinating disease can occur in the apparent absence of neuroantigen-specific autoimmune responses. The relationship of the present results to the immunopathology of multiple sclerosis is discussed.

T cell sensitization to proteolipid protein in myelin basic protein-induced relapsing experimental allergic encephalomyelitis.

Abstract (SJL/J × PL/J)F1 mice immunized with myelin basic protein (MBP) develop an autoimmune demyelinating disease termed relapsing experimental allergic encephalomyelitis (rEAE). The acute stage of disease is mediated by CD4+ T cells specific for MBP amino acids 1–9. To determine the immunologic bases for disease relapse, host sensitization to additional autoantigens of the central nervous system was measured. Results indicate that most animals develop T cell reactivity to endogenous myelin proteolipid protein (PLP) during rEAE. However, PLP-specific immunity does not appear to accound for expression of relapse episodes of demyelination. relapse episodes of demyelination.

Myelin proteolipid protein induces demyelinating disease in mice

Using two methods of immunization (A and B), 5/10 (A) and 27/45 (B), BALB/c by J mice immunized with human myelin proteolipid protein developed a demyelinating disease with a spectrum of chronic progressive to relapsing-remitting courses. Demyelinative lesions were seen histopathologically in all clinically affected animals that were examined. Many of the clinically unaffected animals also had histopathologic evidence of demyelination. Some of the animals had evidence of multiple ages of foci of activity. Evidence is presented that contamination with myelin basic protein could not account for the disease. This is a new model for multiple sclerosis in mice.

Identification of a novel T cell epitope of human proteolipid protein (residues 40-60) recognized by proliferative and cytolytic CD4+ T cells from multiple sclerosis patients

Research into the pathogenesis of multiple sclerosis (MS) has focused on myelin antigens as potential targets of autoimmune attack. Proteolipid protein (PLP) is the most abundant myelin protein comprising more than 50% of central nervous system myelin. Although PLP is a hydrophobic membrane protein which has made it difficult to study, the use of synthetic peptides based on the PLP sequence provides an alternative method for studying the immunological properties of PLP. Using peripheral blood lymphocytes from MS patients, long-term TCL established in the presence of PLP reacted weakly to PLP in proliferation assays; however, these same lines were much more reactive to synthetic peptides of PLP. Thus, we established short-term T cell lines (TCL) from the peripheral blood lymphocytes (PBL) of MS patients in the presence of five separate synthetic PLP peptides. In 6/7 MS patients, proliferative responses were elicited most often to PLP 40-60 compared to four other PLP peptides (PLP 89-106, 103-120, 125-143, and 139-154) (Pelfrey et al., 1993). Interestingly, however, the magnitude of the proliferative response was greatest in response to PLP 89-106. Characterization of PLP 89-106-responsive TCL from several MS patients, indicated that TCL proliferating to the peptide also lysed PLP 89-106 pulsed autologous targets. The majority of cytolytic PLP 89-106 TCL were CD4+ and MHC class II restricted and the predominant restriction elements were those most commonly found in MS patients. These findings suggest that the use of synthetic peptides represents a viable alternative approach to the study of PLP reactivity in humans. We report here that MS PBL recognize several PLP peptides, with the predominant responses to PLP 40-60 and PLP 89-106. Since these cells have both helper (CD4+) and cytolytic capabilities, it is possible that they may play a role in the pathogenesis or progression of MS.

T cell recognition of myelin proteolipid protein and myelin proteolipid protein peptides in the peripheral blood of multiple sclerosis and control subjects.

Myelin proteolipid protein (PLP) is a prime candidate autoantigen for multiple sclerosis. In order to define potential immunodominant epitopes, T cell lines (TCL) from the peripheral blood of HLA-DR 15(2) MS patients were established which responded to the intact molecule of PLP. These TCL were then tested in individual proliferation assays with a variety of PLP peptides spanning most of the PLP molecule. Multiple peptides were recognized by TCL from the MS population, with more than one peptide often recognized by lines from the same individual. Three immunodominant peptides were identified which were recognized by the majority of MS patients. Estimated frequency analyses were then performed on the peripheral blood of HLA-DR15(2)-positive MS and control subjects using TCL initiated by the three immunodominant peptides, 40-60, 95-117, and 185-206. TCL from HLA-DR15 MS subjects recognized peptide 95-117 significantly more often than TCL from control subjects.

Prolonged effects of large-dose methylprednisolone infusion in multiple sclerosis.

Three daily 1-gm doses of methylprednisolone were administered to 12 patients with multiple sclerosis. Granulocytes, lymphocytes, T and B lymphocytes, lymphocyte transformation in response to several mitogens and one antigen, plasma cortisol, and serum IgM and IgA all returned to baseline within a few days. However, total white blood count, unstimulated lymphocyte incorporation of 3H-thymidine, hematocrit, serum IgM, and cerebrospinal fluid IgG synthesis remained altered 1 week after the infusion. The relationship of these changes to any clinical effects is unknown.