Kenneth E. Hill

Platelets Contribute to the Pathogenesis of Experimental Autoimmune Encephalomyelitis

Rationale: Multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE), are inflammatory disorders of the central nervous system (CNS). The function of platelets in inflammatory and autoimmune pathologies is thus far poorly defined. Objective: We addressed the role of platelets in mediating CNS inflammation in EAE. Methods and Results: We found that platelets were present in human MS lesions as well as in the CNS of mice subjected to EAE but not in the CNS from control nondiseased mice. Platelet depletion at the effector-inflammatory phase of EAE in mice resulted in significantly ameliorated disease development and progression. EAE suppression on platelet depletion was associated with reduced recruitment of leukocytes to the inflamed CNS, as assessed by intravital microscopy, and with a blunted inflammatory response. The platelet-specific receptor glycoprotein Ib&agr; (GPIb&agr;) promotes both platelet adhesion and inflammatory actions of platelets and targeting of GPIb&agr; attenuated EAE in mice. Moreover, targeting another platelet adhesion receptor, glycoprotein IIb/IIIa (GPIIb/IIIa), also reduced EAE severity in mice. Conclusions: Platelets contribute to the pathogenesis of EAE by promoting CNS inflammation. Targeting platelets may therefore represent an important new therapeutic approach for MS treatment.

Augmentation of adoptively transferred experimental allergic encephalomyelitis by administration of a monoclonal antibody specific for LFA-1α

We investigated the effect of an anti-leukocyte function antigen 1 (LFA-1 alpha) monoclonal antibody, M17/4.2, on murine relapsing experimental allergic encephalomyelitis (EAE). In vitro investigations demonstrated that M17/4.2 inhibited proliferation with concanavalin A or myelin basic protein. Control mice treated with phosphate buffered saline (PBS) developed a mild to moderate disease at 7-10 days followed by a long-term relapsing clinical course. With administration of M17/4.2, the time of disease onset was unchanged; however, the severity of the disease was greatly augmented, resulting in early mortality. The pathology correlated well with the clinical course. M17/4.2 mice showed more inflammation and demyelination than PBS or anti-CD4 treated mice. Therefore, this anti-LFA-1 specific monoclonal antibody augmented EAE.

Nitric oxide synthase inhibitor, aminoguanidine, reduces inflammation and demyelination produced by Theiler's virus infection

This study evaluated effects of the inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AG), on the neuropathology and clinical disease produced by Theilers murine encephalomyelitis virus (TMEV) DA strain infection. Treatment with AG was started on day 7, 14, 28 or 66 post-inoculation and continued for a minimum of 21 days. Inflammation, demyelination and axonal necrosis were scored in a blinded fashion on spinal cord sections from each mouse. Reduction in inflammation, demyelination and axonal necrosis was observed in mice treated with AG. Apoptosis within the spinal cord parenchyma and perivascular cuffs was significantly decreased. AG treatment resulted in delayed onset of clinical disease.

Gender variations in early Theiler's virus induced demyelinating disease: differential susceptibility and effects of IL-4, IL-10 and combined IL-4 with IL-10

Theilers murine encephalomyelitis virus (TMEV) induced demyelinating disease, is an animal model of multiple sclerosis (MS). The viral-induced encephalitis is followed by an inflammatory and demyelinating disease. We quantitated the response of female and male mice during the transition from encephalitis to early demyelination. CNS neuropathology and antiviral antibody production were evaluated. Parallel studies were done with anti-inflammatory cytokines IL-4, IL-10 or a combination of IL-4 with IL-10. Results show female mice demonstrate an augmented susceptibility to the virus and a greater response to the cytokine therapies. Significant variation was noted during early demyelinating disease. The combination therapy of IL-4 with IL-10 produced striking decreases in antiviral antibody levels and virus-induced neuropathologic disease. Male mice are less susceptible to viral-induced disease and are less responsive to the cytokine treatments. Gender bias in TMEV-induced demyelinating disease appears to parallel the differences noted with other experimental immune diseases.

Quantitation of elastin production in cultured vascular smooth muscle cells by a sensitive and specific enzyme-linked immunoassay.

An enzyme-linked immunoassay (ELISA) procedure has been developed to quantitate the amount of elastin produced by cultured porcine aortic smooth muscle cells. ELISA was used to determine both titer and specificity of antisera raised in rabbits against porcine aortic alpha-elastin conjugated with key-hole limpet hemocyanin. Under optimum conditions (1: 3000 dilution of antiserum, 20 ng alpha-elastin per assay well), sensitivity averaged 60 ng/ml). Specificity was confirmed by immunoprecipitation of [125I]-tropoelastin, radial immunodiffusion, Western blotting and lack of cross-reactivity with serum proteins or collagen. Extensive cross-reactivity was found with both human alpha-elastin and porcine beta-elastin, while porcine tropoelastin was able to compete with 80% of the alpha-elastin determinants. Affinity of anti-porcine antisera for sheep alpha- elastin was significantly lower. When the ELISA was made specific for tropoelastin by coating wells with 60 ng of this antigen, a time-dependent and serum-dependent rate of production of tropoelastin was observed in the culture medium of primary and secondary cultures of smooth muscle cells. Comparison of elastin production in cultures of porcine smooth muscle cells suggests that porcine aortic elastin production varies as a function of cell density and phase of growth.

The pathologic role for COX-2 in apoptotic oligodendrocytes in virus induced demyelinating disease: Implications for multiple sclerosis

The objective of this study was examine whether the inducible isoform of the enzyme cyclooxygenase (COX-2) was expressed in dying oligodendrocytes in the Theilers murine encephalomyelitis virus (TMEV) induced demyelinating disease (TMEV-IDD), an experimental animal model for multiple sclerosis (MS). COX-2 is an enzyme that is tightly coupled to neuronal excitotoxic death. Since neuronal expression of COX-2 contributes to the susceptibility of neurons to excitotoxic death, we asked whether COX-2 was expressed in oligodendrocytes in MS plaques and in lesions during TMEV-IDD. COX-2 was expressed in oligodendrocytes in chronic active lesions from two MS patients. Similar pathology was present in TMEV-IDD spinal cord lesions. COX-2 was expressed in oligodendrocytes four weeks after infection with TMEV coincident with the onset of demyelination. A marker for apoptotic death, activated caspase 3, was present in a subset of oligodendrocytes that expressed COX-2. Oligodendrocyte expression of COX-2 in TMEV-IDD and MS lesions is consistent with a pathological role for this enzyme in demyelination. The presence of the cell death marker (activated caspase 3) with COX-2 in oligodendrocytes is direct evidence linking COX-2 with cell death of oligodendrocytes in these demyelinating diseases.

Microglial inhibition of neuroprotection by antagonists of the EP1 prostaglandin E2 receptor

BackgroundThe EP1 receptor for the prostanoid PGE2 is a G-protein coupled receptor that has been shown to contribute to excitotoxic neuronal death. In this study we examined the influence of non-neuronal cells on neuroprotective properties of EP1 receptor antagonists (Ono 8711 and SC 51089).MethodsPrimary neuronal cultures systems with or without non-neuronal cells were used to examine how the neuroprotective properties of EP1 antagonists were influenced by non-neuronal cells. The influence of astrocytes or microglia were individually tested in excitotoxicity assays using a co-culture system with these cells grown on permeable transwell inserts above the neuronal-enriched cultures. The influence of microglia on PGE2 synthesis and EP1 receptor expression was examined.ResultsEP1 antagonists were neuroprotective in neuronal-enriched cultures (> 90% neurons) but not in mixed cultures (30% neurons plus other non-neuronal cells). Co-cultures of microglia on permeable transwell inserts above neuronal-enriched cultures blocked neuroprotection by EP1 antagonists. Incubation of microglia with neuronal-enriched cultures for 48 hours prior to NMDA challenge was sufficient to block neuroprotection by EP1 antagonists. The loss of neuroprotection by EP1 antagonists was accompanied by a decrease of neuronal EP1 expression in the nucleus in cultures with microglia present.ConclusionThese findings demonstrate microglial modulation of neuronal excitotoxicity through interaction with the EP1 receptor and may have important implications in vivo where microglia are associated with neuronal injury.

Cerebellar Purkinje cells incorporate immunoglobulins and immunotoxins in vitro: implications for human neurological disease and immunotherapeutics

BackgroundImmunoglobulin G (IgG) antibodies reactive with intracellular neuronal proteins have been described in paraneoplastic and other autoimmune disorders. Because neurons have been thought impermeable to immunoglobulins, however, such antibodies have been considered unable to enter neurons and bind to their specific antigens during life. Cerebellar Purkinje cells - an important target in paraneoplastic and other autoimmune diseases - have been shown in experimental animals to incorporate a number of molecules from cerebrospinal fluid. IgG has also been detected in Purkinje cells studied post mortem. Despite the possible significance of these findings for human disease, immunoglobulin uptake by Purkinje cells has not been demonstrated in living tissue or studied systematically.MethodsTo assess Purkinje cell uptake of immunoglobulins, organotypic cultures of rat cerebellum incubated with rat IgGs, human IgG, fluorescein-conjugated IgG, and rat IgM were studied by confocal microscopy in real time and following fixation. An IgG-daunorubicin immunotoxin was used to determine whether conjugation of pharmacological agents to IgG could be used to achieve Purkinje cell-specific drug delivery.ResultsIgG uptake was detected in Purkinje cell processes after 4 hours of incubation and in Purkinje cell cytoplasm and nuclei by 24-48 hours. Uptake could be followed in real time using IgG-fluorochrome conjugates. Purkinje cells also incorporated IgM. Intracellular immunoglobulin did not affect Purkinje cell viability, and Purkinje cells cleared intracellular IgG or IgM within 24-48 hours after transfer to media lacking immunoglobulins. The IgG-daunomycin immunotoxin was also rapidly incorporated into Purkinje cells and caused extensive, cell-specific death within 8 hours. Purkinje cell death was not produced by unconjugated daunorubicin or control IgG.ConclusionPurkinje cells in rat organotypic cultures incorporate and clear host (rat) and non-host (human or donkey) IgG or IgM, independent of the immunoglobulins reactivity with Purkinje cell antigens. This property permits real-time study of immunoglobulin-Purkinje cell interaction using fluorochrome IgG conjugates, and can allow Purkinje cell-specific delivery of IgG-conjugated pharmacological agents. Antibodies to intracellular Purkinje cell proteins could potentially be incorporated intracellularly to produce cell injury. Antibodies used therapeutically, including immunotoxins, may also be taken up and cause Purkinje cell injury, even if they do not recognize Purkinje cell antigens.

Using Diffusion Tensor Imaging and Immunofluorescent assay to evaluate the pathology of Multiple Sclerosis

To determine the ability of the principal diffusion tensor imaging (DTI) indices to predict the underlying histopathology evaluated with immunofluorescent assay (IFA).

Chimeric cytotoxin IL2-PE40 inhibits relapsing experimental allergic encephalomyelitis

IL2-PE40 is a chimeric protein composed of human interleukin-2 (IL2) genetically fused to a modified form of Pseudomonas exotoxin lacking the cell recognition domain. IL2-PE40 is cytotoxic for IL2 receptor-bearing lymphocytes in culture and can inhibit activation of T cells in vivo. IL2-PE40 can significantly diminish antigen-stimulated proliferation of lymphocytes sensitized to myelin basic protein. Intraperitoneal administration of IL2-PE40 not only markedly inhibits the clinical manifestations of adoptively transferred relapsing experimental allergic encephalomyelitis but also dramatically reduces both inflammation and demyelination characteristic of the disease.