Il-Kwon Park

CDR3 Spectratyping Analysis of the TCR Repertoire in Myasthenia Gravis

Because myasthenia gravis (MG) is an autoimmune disease mediated by Abs specific for the acetylcholine receptor, helper T cells play a role in Ab production. In this study, we have performed large-scale cross-sectional and longitudinal TCR studies by CDR3 spectratyping using PBL and thymus tissues from MG patients. We found that there was no preferential usage of any particular TCR β-chains that was identical among MG patients. However, the longitudinal study clearly demonstrated that one or more TCR Vβ expansions persisted frequently in MG patients. Importantly, persistent TCR expansions correlated with clinical severity and high anti-acetylcholine receptor Ab titer. Finally, examinations of T cells expressing CXCR5, i.e., follicular B-helper T cells, revealed that spectratype expansions in MG patients were detected mainly in the CD4+ CXCR5+ T cell populations, whereas CD8+ T cells were the major source of clonal expansion in healthy subjects. These findings suggest that persistent clonal expansions of T cells in MG patients are associated with the development and maintenance of MG. Close examination of pathogenic T cells in MG provides useful information to elucidate the pathogenesis and to estimate the disease status.

Matrix Metalloproteinase (MMP)-9, but Not MMP-2, Is Involved in the Development and Progression of C Protein-Induced Myocarditis and Subsequent Dilated Cardiomyopathy

Repeated or continuous inflammation of the heart is one of the initiation factors for dilated cardiomyopathy (DCM). In previous studies, we established a DCM animal model by immunizing rats with cardiac C protein. In the present study, we analyze the role of matrix metalloproteinases (MMPs) in experimental autoimmune carditis (EAC) and subsequent DCM to elucidate the pathomechanisms of this disease. In this model, inflammation begins ∼9 days after immunization. At that time, MMP activities were detected by in situ zymography. Real-time PCR analysis revealed continuous up-regulation of MMP-2 mRNA from 2 wk and thereafter. MMP-9 mRNA, however, had only a transient increase at 2 wk. Double staining with in situ zymography and cell markers demonstrated that gelatinase (MMP-2 and MMP-9)-expressing cells are infiltrating macrophages during the early stage and cardiomyocytes at later stages. Minocycline, which inhibits MMP-9 activities more strongly than MMP-2, significantly suppressed EAC, but an MMP-2-specific inhibitor, TISAM, did not affect the course of the disease. Furthermore, immunohistochemical examination revealed that minocycline treatment suppressed T cell and macrophage infiltration strongly, whereas TISAM did not. These findings indicate that MMP-9, but not MMP-2, is involved in the pathogenesis of the acute phase of EAC, and further suggest that MMP-9 inhibitors, minocycline and its derivatives, may be useful therapies for EAC and DCM.

Paralysis of CD4+CD25+ regulatory T cell response in chronic autoimmune encephalomyelitis

Increasing evidence strongly suggest that CD4(+)CD25(+) regulatory T (Treg) cells play a pivotal role in suppressing the development of autoimmune diseases. However, it remains poorly understood how these cells are involved in the persistence of, or recovery from, the diseases. In the present study, we examined the role of CD4(+)CD25(+) Treg cells in chronic EAE and compared the results with those obtained in acute EAE. In EAE lesions, CD25(+) cells decreased rapidly at the beginning of chronic EAE, whereas these cells were maintained at high levels during the recovery from acute EAE. The number of Foxp3(+)CD4(+)CD25(+) Treg and levels of Foxp3 mRNA in the lymphoid organ were significantly lower in chronic EAE. Importantly, the regulatory function of individual CD4(+)CD25(+) Treg cells was maintained in animals with chronic EAE. Furthermore, adoptive transfer of activated CD4(+)CD25(+) Treg cells suppressed the development of chronic EAE. These findings suggest that impairment of the CD4(+)CD25(+) Treg response is critical for development of chronic autoimmune diseases, and can be adjustable by autologous Treg transplantation.

Characterization of relapsing autoimmune encephalomyelitis and its treatment with decoy chemokine receptor genes

To elucidate the pathomechanisms of relapses of autoimmune disorders and to develop immunotherapy against relapses, we induced acute monophasic and chronic relapsing (CR) experimental autoimmune encephalomyelitis (EAE) in DA rats. Immunopathological and cytokine-chemokine analyses demonstrated that the number of infiltrating macrophages was significantly elevated in the CR-EAE than in acute EAE lesions and that IFN-gamma and IP-10 in the spinal cord were significantly upregulated during the first attack and relapse of CR-EAE, respectively, than at the peak of acute EAE. In vivo administration of decoy chemokine receptor plasmid DNAs encoding the binding sites of CXCR3 and CCR2 suppressed the development of relapse of CR-EAE. Importantly, multiple injections of DNAs did not elicit the antibody production against chemokine receptors. Taken together, these findings demonstrated that neutralization therapy with decoy chemokine receptor DNAs is effective to control autoimmune diseases.

Nonviral DNA Vaccination Augments Microglial Phagocytosis of β-Amyloid Deposits as a Major Clearance Pathway in an Alzheimer Disease Mouse Model

Immunotherapies markedly reduce &bgr;-amyloid (A&bgr;) burden and reverse behavioral impairment in mouse models of Alzheimer disease. We previously showed that new A&bgr; DNA vaccines reduced A&bgr; deposits in Alzheimer disease model mice without detectable side effects. Although they are effective, the mechanisms of A&bgr; reduction by the DNA vaccines remain to be elucidated. Here, we analyzed vaccinated and control Alzheimer disease model mice from 4 months to 15 months of age to assess which of several proposed mechanisms may underlie the beneficial effects of this vaccination. Immunohistochemical analysis revealed that activated microglial numbers increased significantly in the brains of vaccinated mice after DNA vaccination both around A&bgr; plaques and in areas remote from them. Microglia in treated mice phagocytosed A&bgr; debris more frequently than they did in untreated mice. Although microglia had an activated morphological phenotype, they did not produce significant amounts of tumor necrosis factor. Amyloid plaque immunoreactivity and A&bgr; concentrations in plasma increased slightly in vaccinated mice compared with controls at 9 but not at 15 months of age. Collectively, these data suggest that phagocytosis of A&bgr; deposits by microglia plays a central role in A&bgr; reduction after DNA vaccination.

Differential effects of decoy chemokine (7ND) gene therapy on acute, biphasic and chronic autoimmune encephalomyelitis: Implication for pathomechanisms of lesion formation

Multiple sclerosis (MS) exhibits several clinical subtypes such as the relapsing-remitting (RR) and secondary progressive (SP) forms. In accordance with this, formation of demyelinating plaques in the central nervous system (CNS) occurs by different mechanisms. In the present study, we induced acute, biphasic and chronic (RR or SP) EAE in rats and examined the effects of decoy chemokine (7ND) gene therapy, which inhibits the migration of macrophages, to address the above issue. Interestingly, it was demonstrated that the clinical signs of acute EAE and the first attack of biphasic EAE were minimally affected, whereas chronic EAE and the relapse of biphasic EAE were completely suppressed with 7ND treatment. In the CNS, the number of infiltrating macrophages was reduced in all the stages of the three types of EAE. These findings suggest that in acute EAE and in the first attack of biphasic EAE, where anti-macrophage migration therapy was almost ineffective, pathogenic T cells are mainly involved in lesion formation. In contrast, the relapse of biphasic EAE and chronic EAE macrophages play a major role in the disease process. Thus, the mechanisms of lesion formation are not uniform and immunotherapy should be performed on the basis of information about the pathomechanisms of autoimmune diseases.

Characterization of CD8‐positive macrophages infiltrating the central nervous system of rats with chronic autoimmune encephalomyelitis

CD8+ macrophages appear in the central nervous system (CNS) under various pathological conditions such as trauma and ischemia. Furthermore, macrophages expressing CD8 were found in CNS lesions of chronic, but not acute, experimental autoimmune encephalomyelitis (EAE). To further characterize cells with this phenotype, we examined CD8+ macrophages/monocytes in the CNS and peripheral organs during the course of acute and chronic EAE that had been induced by immunization of rats with myelin basic protein and myelin oligodendrocyte glycoprotein, respectively. Counting CD8+ macrophages in CNS lesions revealed that their numbers increased reaching about 60% of total infiltrating macrophages in chronic EAE, while CD8+ macrophages remained less than 5% throughout the course of acute EAE. Unexpectedly, however, higher abundance of CD8+ monocytes/macrophages in the peripheral blood was found in both acute and chronic EAE. Real‐time polymerase chain reaction analysis revealed no significant difference in the levels of chemokines and chemokine receptors of blood CD8+ monocytes between acute and chronic EAE. mRNA expression of perforin, a cytotoxic substance, was up‐regulated in CD8+ monocytes compared with that of CD8− monocytes in both acute and chronic EAE. These findings suggest that activated CD8+ macrophages may play a cytotoxic role in chronic EAE lesions and that cells other than CD8+ monocytes/macrophages determined the difference in CNS pathology between acute and chronic EAE. Analysis of CD8+ monocytes/macrophages may provide useful information to permit further dissect the pathomechanisms of multiple sclerosis and to develop effective immunotherapies against autoimmune diseases in the CNS.

Characterization of pathogenic T cells and autoantibodies in C-protein-induced autoimmune polymyositis

Although autoimmune processes may take place in human polymyositis, little is known with regard to its pathogenesis due to the lack of appropriate animal models. In the present study, we developed experimental autoimmune myositis (EAM) in Lewis rats by immunization with recombinant skeletal C-protein and examined the role of pathogenic T cells and autoantibodies. Using recombinant proteins and synthetic peptides, we demonstrated that skeletal C-protein Fragment 2 (SC2) has the strongest myositis-inducing ability and that myositis-inducing epitope(s) reside within the residues 334-363 of SC2 (SC2P3). However, immunization with SC2P3 induced only mild EAM compared with SC2 immunization. Characterization of T cells and antisera revealed that SC2P3 and SC2P7 contain the B cell epitope, while the T cell epitope resides in SC2P5. Furthermore, anti-SC2, but not anti-SC2P3, antisera contained antibodies against the conformational epitope(s) in the SC2 molecule. However, SC2P3 or SC2P5 immunization plus anti-SC2 antibody transfer aggravated the disease only slightly. These findings suggest that C-protein-induced EAM is formed by activation of C-protein-specific T cells along with antibodies against conformational epitopes in C-protein but that there are undetermined factors related to the disease progression. Further analysis of C-protein-induced EAM will provide useful information to elucidate the pathomechanisms of human polymyositis.

Quantitation of myelin oligodendrocyte glycoprotein and myelin basic protein in the thymus and central nervous system and its relationship to the clinicopathologic features of autoimmune encephalomyelitis

There is controversy whether the amount of autoantigens expressed in the thymus regulates negative selection of autoreactive T cells and determine susceptibility or resistance to experimental autoimmune encephalomyelitis (EAE). In the present study, we have addressed this issue by quantifying neuroantigens in the thymus of two EAE‐susceptible (LEW and LEW.1AV1) and one EAE‐resistant (BN) rat strains. We further examined whether amounts of neuroantigens in various parts of the central nervous system (CNS) affect the clinical course and lesion distribution of acute and chronic EAE. Real‐time PCR and histologic analyses showed that there was no significant difference in the amount and distribution of myelin oligodendrocyte glycoprotein and myelin basic protein in the thymus and CNS among the three strains and that both acute and chronic EAE lesions in the CNS were preferentially distributed in the area where neuroantigens were abundantly present. These findings suggest that susceptibility or resistance to EAE is not regulated by the amount of the neuroantigens expressed in the thymus. Furthermore, the lesion distribution, but not the clinical course, of EAE is related to the neuroantigen expression in the CNS.