Kazuya Iwabuchi

Amelioration of experimental autoimmune encephalomyelitis in C57BL/6 mice by an agonist of peroxisome proliferator-activated receptor-γ

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a member of the nuclear hormone receptor superfamily, plays a critical role in adipocyte differentiation and glucose homeostasis. It has been implicated that PPAR-gamma functions as a regulator of cellular proliferation and inflammatory responses. In the present study, we examined whether troglitazone, a selective PPAR-gamma agonists, ameliorated experimental autoimmune encephalomyelitis (EAE) induced by administration of myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 in C57BL/6 mice. We found that troglitazone attenuated the inflammation and decreased the clinical symptoms. It was suggested that the amelioration was attributed to the attenuation of pro-inflammatory cytokine gene expressions.

Activation of extracellular signal-related kinase by TNF-α controls the maturation and function of murine dendritic cells

Functional roles of extracellular signal‐related kinase (ERK)activation in dendritic‐cell (DC) maturation have been unclear. In thepresent study, we investigated the ERK pathway in tumor necrosis factor(TNF)‐α‐induced maturation of murine spleen‐derived DC. TNF‐αincreased surface expressions of major histocompatibility(MHC) and costimulatory molecules on DC in a dose‐dependentmanner. High (40 ng/ml) and low (0.4 ng/ml) concentrations of TNF‐αmarkedly enhanced ERK1/2 activation in DC, and this activation wasblocked completely by PD98059, a selective inhibitor of the ERKpathway. When DC were treated with TNF‐α at a low but not a highconcentration, PD98059 notably enhanced surface expressions of the MHCand costimulatory molecules and allostimulatory capability of the DC. Interleukin (IL)‐12 production was enhanced significantly by PD98059 inDC treated with low or high concentration of TNF‐α. These findingssuggest that TNF‐α‐induced ERK activation negatively controlsmaturation and IL‐12 production in murine DC.

Induction of CTL Responses by Simultaneous Administration of Liposomal Peptide Vaccine with Anti-CD40 and Anti-CTLA-4 mAb

Activation of APC via CD40-CD40 ligand pathway induces up-regulation of costimulatory molecules such as B7 and production of IL-12. Interaction between B7 on APC and CD28 on naive T cells is necessary for priming the T cells. On the other hand, interaction between B7 on APC and CTLA-4 on activated T cells transduces a negative regulatory signal to the activated T cells. In the present study, we attempted to generate tumor-specific CTL by s.c. administration of antigenic peptides encapsulated in multilamellar liposomes (liposomal peptide vaccine) with anti-CD40 mAb and/or anti-CTLA-4 mAb. Liposomal OVA257–264 and anti-CD40 mAb or anti-CTLA-4 mAb were administrated to C57BL/6 mice and the splenocytes were cocultured with OVA257–264 for 4 days. The splenic CD8+ T cells showed a significant cytotoxicity against EL4 cells transfected with cDNA of OVA. In addition, administration of both anti-CD40 and anti-CTLA-4 mAb enhanced the CTL responses. Considerable CTL responses were induced in MHC class II deficient mice by the same procedure. This finding indicated that CTL responses could be generated even in the absence of Th cells. When BALB/c mice were immunized with pRL1a peptide that are tumor-associated Ag of RL♂1 leukemia cells using the same procedure, significant CTL responses were induced and prolonged survival of the BALB/c mice was observed following RL♂1 inoculation. These results demonstrate that anti-CD40 mAb and anti-CTLA-4 mAb function as immunomodulators and may be applicable to specific cancer immunotherapy with antitumor peptide vaccine.

Identification of a peptide inducing experimental autoimmune uveoretinitis (EAU) in H-2Ak-carrying mice

When certain strains of mice bearing H‐2Ak are immunized with the interphotoreceptor retinoid‐binding protein (IRBP), EAU is induced. Thus far uveitogenic determinant(s) has not been determined in the H‐2Ak mouse system. In addition it is hard to prepare purified IRBP. In the present study, to circumvent these problems we attempted to identify uveitogenic peptides derived from bovine IRBP in H‐2Ak haplotype mice. Six peptides which had been selected according to the H‐2Ak binding motif (Dxxxxxxxx[A, R, T]) were synthesized. We report here that all the peptides are immunogenic but only one peptide, K2, which consisted of IRBP201–216 residues, induces EAU in various mice carrying H‐2Ak. Amino acid substitution of K2 revealed that the core region interacted with both H‐2Ak and T cell antigen receptor (TCR). The amino acid sequence of the core region derived from bovine IRBP was identical to the corresponding region of mouse IRBP. In addition, K2 appeared to be a natural peptide antigen processed from bovine IRBP. Altogether, we concluded that K2 is one of the natural autoantigens involved in induction of EAU in H‐2Ak mice.

Enhancement of stromal cell-derived factor-1α-induced chemotaxis for CD4/8 double-positive thymocytes by fibronectin and laminin in mice

Stromal cell‐derived factor‐1α (SDF‐1α) is a chemokine abundantly expressed in the thymus. However, a potential role of SDF‐1α in the thymus has been under consideration, since no appreciable difference was detected in the migratory responsiveness to the SDF‐1α between cortical and medullary thymocytes. In the present study, we examined the effects of extracellular matrix (ECM) on the responsiveness of murine thymocytes to several chemokines including SDF‐1α. In the absence of ECM, chemotactic activity of SDF‐1α for cortical (CD4/8 double‐positive) thymocytes was almost same as that for medullary (CD4 or CD8 single‐positive) thymocytes. In contrast, the chemotactic activity of SDF‐1α for cortical thymocytes was considerably (more than 10‐fold) enhanced by laminin or fibronectin as compared with that for medullary thymocytes. Chemotactic activities of macrophage‐derived chemokine and macrophage inflammatory protein‐3β for both cortical and medullary thymocytes were only slightly enhanced by fibronectin or laminin. Thus, fibronectin and laminin appear to enhance the chemotactic activity of SDF‐1α for cortical thymocytes selectively. Addition of a monoclonal antibody against CD29 showed no inhibitory effect on the enhanced chemotactic activity of SDF‐1α, suggesting that the other unknown receptor(s) is involved in this enhancement. Our present data demonstrate that SDF‐1α in the presence of fibronectin or laminin is involved in the distribution of developing thymocytes.

Serum level of macrophage migration inhibitory factor as a useful parameter of clinical course in patients with Wegener's granulomatosis and relapsing polychondritis.

Novel biological activities of macrophage migration inhibitory factor (MIF) have been rediscovered. In addition, elevation of the serum MIF level has been reported in different types of disorders, including various inflammatory and autoimmune diseases. In the present study, serum MIF levels were analyzed in patients with Wegeners granulomatosis (WG) and relapsing polychondritis. It was shown that the serum MIF levels in these patients were significantly higher than those of normal healthy controls. In a WG patient, the MIF level showed a good correlation with clinical symptoms and C-ANCA titers. Thus, serum MIF levels will be a useful laboratory parameter for following the clinical course of WG patients and determining medical treatment. The immunopathologic roles of MIF in these diseases are discussed.

Antisense Macrophage Migration Inhibitory Factor (MIF) Prevents Anti-IgM Mediated Growth Arrest and Apoptosis of a Murine B Cell Line by Regulating Cell Cycle Progression

Macrophage migration inhibitory factor (MIF) is involved in the generation of cell‐mediated immune responses. Recently it has been reported that MIF also plays a role in cell proliferation and differentiation. In the present study, using a B‐cell line, WEHI‐231, and its stable MIF‐antisense transfectant, WaM2, as a representative transfectant, we investigated the mechanism underlying regulation of the cell growth by MIF. WaM2 cells produced less MIF than vector control or parental WEHI‐231 cells. Reduced and increased proportions were seen in G1 and S‐phase cells, respectively, in WaM2 as compared with WEHI‐231. Growth arrest and apoptosis after stimulation via surface Ig (sIg) were less prominent in WaM2 cells than those in WEHI‐231. However, the addition of recombinant rat MIF did not reverse the inhibition of the growth arrest and apoptosis induced in WaM2 by cross‐linking sIg. Almost the same amount of p27kipI expression was detected in WaM2 cells as those in WEHI‐231 and vector control cells. Cross‐linking of sIg elevated the p27kipI level equally in these cells irrespective of the MIF‐antisense expression. Taken together, it seems that MIF plays a role in inducing apoptosis in B cells upon IgM cross‐linking by regulating the cell cycle via a novel intracellular pathway.

Different influence of macrophage migration inhibitory factor (MIF) in signal transduction pathway of various T cell subsets.

It has been shown that macrophage migration inhibitory factor (MIF) modulates not only macrophage functions, but also T cell functions. However, detailed analysis of the MIF function on responses of various T cell subpopulations remained to be elucidated. In this report, using a neutralizing anti-MIF monoclonal antibody (mAb) we examined MIF functions on various T cell lineages. It was shown that anti-MIF mAb inhibited antigen-specific responses of both IFN-gamma producing and IL-4 producing T cells. The inhibition appeared to be related to blockade of the signal pathway via T cell receptor (TCR) but not that via IL-2 receptor (IL-2R). However, the anti-MIF mAb showed no inhibitory effect on NK-T cell responses stimulated through TCR. These results suggest that MIF is involved in the signal pathway via TCR in mainstream T cells but not in NK-T cells.

Abrogation of negative selection by GVHR induced by minor histocompatibility antigens or H-2D antigen alone.

Allogeneic bone marrow chimeras were prepared by donor and recipient combinations that differed in minor histocompatibility loci or H-2D locus alone. When 1 x 10(5) splenic T cells were inoculated in addition to T cell-depleted bone marrow cells (1 x 10(7)), clinically detectable GVHR was induced. In these GVHR chimeras, substantial numbers of T cells reactive to either donor or recipient antigens were both phenotypically and functionally detected. The mechanisms underlying the abrogation of intrathymic negative selection are discussed.

Induction of Endometriosis and Adenomyosis by Transvaginal Pituitary Transplantation in Mice with and without Natural Killer Cell Activity

PROBLEM: The aims of this study were to establish a mouse model of endometriosis and adenomyosis and to elucidate the necessity of reduced natural killer (NK)‐cell and T‐cell activities in the establishment of endometriosis and adenomyosis.