ABSTRACT
We earlier found that a rat monoclonal antibody (mAb) RE2 can induce rapid death of murine activated, but not resting, lymphocytes and lymphocyte cell lines, in a complement-independent manner, a cell death differing from typical apoptosis or necrosis. We here found that this cell death is independent of pathways involving Fas, caspase, and phosphoinositide-3 kinase. With the advantage of producing human B cell line transfectants with stable expression of human/mouse xeno-chimeric MHC class I genes, we found that RE2 epitope resides on the murine class I alpha2 domain. However, the alpha3 domain plays a key role in transducing the death signal, which mediates extensive aggregation of the MHC class I-integrin-actin filament system, giving rise to membrane blebs and pores. In mouse models with T/NKT cell activation-associated fulminant hepatitis, administration of mAb RE2 almost completely inhibited the development of liver cell injuries. Taken collectively, this form of cell death may be involved in homeostatic immune regulation, and induction of this form of cell death using the mAbs may be potentially therapeutic for subjects with immunological diseases mediated by activated lymphocytes.
Subject(s)
Antibodies, Monoclonal/immunology , Cell Death , Hepatitis, Animal/pathology , Histocompatibility Antigens Class I/immunology , Liver/pathology , Lymphocytes/immunology , Animals , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Cell Line , Concanavalin A , Disease Models, Animal , Epitopes , Genes, MHC Class I , Hepatitis, Animal/chemically induced , Hepatitis, Animal/drug therapy , Hepatitis, Animal/immunology , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolism , Humans , Lymphocyte Activation , Lymphocytes/cytology , Major Histocompatibility Complex , Mice , Mice, Inbred C57BL , Protein Structure, Tertiary , Rats , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolismABSTRACT
Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), can be induced by the immunization of mice with myelin antigens in the form of myelin oligodendrocyte glycoprotein (MOG). Macrophage colony-stimulating factor (M-CSF) is required for the development of individual mononuclear phagocyte populations and is involved in the immune response. We previously reported that Ki20227 (N-{4-[(6,7-dimethoxy-4-quinolyl)oxy]-2-methoxyphenyl}-N'-[1-(1,3-thiazole-2-yl)ethyl]urea) is a highly selective M-CSF receptor (c-fms) tyrosine kinase inhibitor. In our current study, we investigated whether Ki20227 has suppressive effects upon EAE and indeed found that this drug significantly reduced the severity of this disease both preventively and therapeutically. Notably also, Ki20227 treatments inhibited the turn-over/expansion of myeloid cells provoked by the immunization and subsequent MOG-specific T cell responses in our EAE animal model. These findings suggest that M-CSF plays a pivotal role in the development of EAE and that Ki20227 and its derivatives may be candidate drugs for the treatment of human MS.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/drug therapy , Phenylurea Compounds/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Thiazoles/therapeutic use , Animals , Antigens, CD/metabolism , Cell Proliferation/drug effects , Cytokines/metabolism , Dose-Response Relationship, Drug , Encephalomyelitis, Autoimmune, Experimental/chemically induced , Encephalomyelitis, Autoimmune, Experimental/pathology , Encephalomyelitis, Autoimmune, Experimental/prevention & control , Flow Cytometry/methods , Lymphocyte Activation/drug effects , Male , Mice , Mice, Inbred C57BL , Myelin Proteins , Myelin-Associated Glycoprotein , Myelin-Oligodendrocyte Glycoprotein , Spinal Cord/pathology , T-Lymphocytes/drug effects , Time FactorsABSTRACT
UNLABELLED: The effectiveness of a new antibody treatment on arthritis-associated osteolysis was studied by using CIA mice. GGT, a newly identified bone-resorbing factor, was upregulated in arthritic joints. We generated monoclonal antibodies against GGT and injected them into CIA mice. Mice treated with antibodies showed a reduction in osteoclast number and bone erosion. INTRODUCTION: Gamma-glutamyl transpeptidase (GGT) acts as a bone-resorbing factor that stimulates osteoclast formation. GGT expression has been detected in active lymphocytes that accumulate at inflammation sites, such as rheumatoid arthritis (RA). We hypothesize that GGT is an effective target for suppression of arthritis-related osteoclastogenesis and joint destruction. Here, we describe the therapeutic effect of neutralizing antibodies against GGT on joint destruction using a collagen-induced arthritis (CIA) mouse model. MATERIALS AND METHODS: GGT expression in the synovium of RA patients and CIA mice was determined by immunohistochemistry and RT-PCR. Monoclonal antibodies were generated against recombinant human GGT (GGT-mAbs) using BALB/c mice. Antibody treatment was performed by intraperitoneal injections of GGT-mAbs into CIA mice. Effects of antibody treatment on arthritis and bone erosion were evaluated by incidence score, arthritis score, and histopathological observations. The role of GGT in osteoclast development was examined by using the established osteoclastogenic culture system. RESULTS: GGT expression was significantly upregulated in inflamed synovium. Immunohistochemistry revealed that GGT was present in lymphocytes, plasma cells, and macrophages, as well as capillaries. Injection of GGT-mAbs significantly decreased the number of osteoclasts and attenuated the severity of joint destruction in CIA mice. In vitro examination showed that GGT enhanced RANKL-dependent osteoclast formation. GGT stimulated the expression of RANKL in osteoblasts and its receptor RANK in osteoclast precursors, respectively. CONCLUSIONS: This study indicates that inflamed synovial tissue-derived GGT acts as a risk factor for joint destruction and that the antibody-mediated inhibition of GGT significantly decreases osteoclast number and bone erosion in CIA mice. GGT antagonists might be novel therapeutic agents for attenuating joint destruction in RA patients.
Subject(s)
Antibodies, Monoclonal/pharmacology , Arthritis, Experimental/enzymology , Cytokines/antagonists & inhibitors , Osteolysis/enzymology , gamma-Glutamyltransferase/antagonists & inhibitors , Animals , Arthritis, Experimental/drug therapy , Arthritis, Experimental/pathology , Cells, Cultured , Cytokines/metabolism , Humans , Inflammation/drug therapy , Inflammation/enzymology , Inflammation/pathology , Lymphocytes/enzymology , Lymphocytes/pathology , Mice , Mice, Inbred BALB C , Osteoblasts/enzymology , Osteoblasts/pathology , Osteoclasts/enzymology , Osteoclasts/metabolism , Osteoclasts/pathology , Osteolysis/drug therapy , Osteolysis/pathology , RANK Ligand/biosynthesis , Rats , Rats, Wistar , Stem Cells/enzymology , Stem Cells/pathology , gamma-Glutamyltransferase/metabolismABSTRACT
BACKGROUND: Germinated barley foodstuff (GBF), which contains glutamine-rich protein and hemicellulose-rich fiber, exhibits therapeutic effects in ulcerative colitis; however, its mechanism is still under investigation. The aim of this study was to evaluate the anti-inflammatory effects of GBF on colitis in terms of the epithelial inflammatory response. METHODS: Mice with dextran sulfate sodium-induced colitis were used. The effects of GBF on the colitis were evaluated by measuring the body weight; disease activity; mucosal damage (histology, mucosal inflammatory parameters, nuclear factor kappa B [NFkB] activation, and signal transducer and activator of transcription 3 [STAT3]); serum interleukin 6 (IL-6) level; cecal short-chain fatty acids (SCFAs); and bile acid contents. RESULTS: GBF significantly prevented disease activity and body weight loss after induction of colitis. Serum IL-6 level and mucosal STAT3 expression were also significantly attenuated, with a conspicuous reduction of mucosal damage; NFkB activity showed the same tendency. Cecal butyrate content was significantly higher and, interestingly, GBF mice had lower bile acid concentrations than the control group. CONCLUSIONS: GBF has the potential to reduce the epithelial inflammatory response by depressing STAT-3 expression and inhibiting NFkB binding activity. These effects may be brought about by an increase of butyrate production and adsorption of bile acids.
Subject(s)
Colitis/diet therapy , Hordeum , Phytotherapy/methods , Plant Preparations/therapeutic use , Animals , Butyrates/metabolism , Colitis/pathology , DNA-Binding Proteins/analysis , Female , Germination , Interleukin-6/blood , Intestinal Mucosa/chemistry , Intestine, Small/microbiology , Mice , Mice, Inbred BALB C , NF-kappa B/analysis , STAT3 Transcription Factor , Trans-Activators/analysisABSTRACT
The purpose of this study was to determine whether species differences in neonatal vocalizations of rodent pups could be observed. Ultrasonic vocalizations of pups of 5 rodent species, mouse (ICR), vole (Microtus arvalis), Syrian hamster, rat (Wistar-Imamichi), and Mongolian gerbil were recorded from 3 to 15 or 21 days of age. Recordings were made under conditions of separation from mothers and litter mates in a cooled chamber (approximately 10 degrees C). The major species differences observed were age specific and species specific frequencies. The Mongolian gerbil displayed a different frequency change with age. Namely, the day on which ultrasonic vocalizations ceased was delayed in Mongolian gerbil compared with the other rodents. The modal peak frequencies of ultrasound emitted from pups at 3 days of age were low (around 35 kHz) in the vole and the Syrian hamster, medium (around 45 kHz) in the rat and the Mongolian gerbil, and high (around 55 kHz) in the mouse.
Subject(s)
Rodentia/physiology , Vocalization, Animal , Animals , Animals, Newborn , Arvicolinae , Cricetinae , Female , Male , Mesocricetus , Mice , Mice, Inbred ICR , Rats , Rats, Wistar , Social Isolation , Species Specificity , UltrasonicsABSTRACT
Macrophage colony-stimulating factor (M-CSF) is important in the development of macrophages and osteoclasts. Previous studies have also shown that CD11b(+) myeloblasts and osteoclasts play key roles during inflammation and bone destruction in arthritic lesions. In this study, we investigated whether N-{4-[(6,7-dimethoxy-4-quinolyl)oxy]-2-methoxyphenyl}-N'-[1-(1,3-thiazole-2-yl)ethyl] urea (Ki20227), an inhibitor of the M-CSF receptor (c-Fms), suppressed disease progression in a type II collagen (CII)-induced arthritis (CIA) mouse model. We found that Ki20227 inhibited M-CSF-dependent reactions, such as lipopolysaccharide-induced tumor necrosis factor-alpha production, which were enhanced by M-CSF in vitro. Oral administration of Ki20227 in vivo prevented inflammatory cell infiltration and bone destruction, and consequently suppressed disease progression. In addition, the number of CD11b(+), Gr-1(+), and Ly-6G(+) cells in the spleen decreased in the Ki20227-treated mice, and the CII-induced cytokine production in splenocytes isolated from the Ki20227-treated arthritic mice was also reduced. These observations indicate that Ki20227 might exert its therapeutic effects in the CIA mouse model by suppressing the M-CSF-dependent accumulation of both inflammatory and osteoclast cells, as well as by inhibiting inflammatory cytokine production. Hence, inhibitors of the c-Fms tyrosine kinase might act as anti-inflammatory or anti-osteolytic agents against arthritis.
Subject(s)
Arthritis, Experimental/drug therapy , Inflammation/drug therapy , Phenylurea Compounds/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Thiazoles/therapeutic use , Animals , Arthritis, Experimental/immunology , Blotting, Western , Bone and Bones/drug effects , Cytokines/biosynthesis , Cytokines/drug effects , Disease Progression , Flow Cytometry , Macrophage Colony-Stimulating Factor/drug effects , Male , Mice , Osteoclasts/drug effects , Spleen/drug effectsABSTRACT
We previously reported that 4C8 monoclonal antibody (mAb) provides a costimulatory signal to human CD4+ T cells and consequently induces regulatory T (Treg) cells, which are hypo-responsive and suppress the polyclonal response of bystander CD4+ cells in a contact-dependent manner. In this study, we identified the antigen of 4C8 mAb as CD52. Costimulation with Campath-1H, a humanized anti-CD52 mAb, also induced Treg cells. Anti-CD52-induced Treg cells suppressed the proliferation of both CD4+ and CD8+ T cells provided with polyclonal or allogeneic stimulation. When Treg cells were induced from Staphylococcal enterotoxin B (SEB) treated cells, they suppressed the response to SEB more efficiently than that to another superantigen, SEA. Furthermore, anti-CD52-induced Treg cells could be expanded by culture with IL-2 followed by CD52-costimulation, and co-injection of expanded Treg cells suppressed lethal xenogeneic graft versus host disease (GvHD) reactions in SCID mice caused by human peripheral blood mononuclear cells (PBMCs).
Subject(s)
Antigens, CD/immunology , Antigens, Neoplasm/immunology , Glycoproteins/immunology , T-Lymphocytes, Regulatory/immunology , Alemtuzumab , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal, Humanized , Antibodies, Neoplasm/immunology , Antigens, CD/biosynthesis , CD52 Antigen , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Enterotoxins/immunology , Epitopes, T-Lymphocyte/immunology , Female , Forkhead Transcription Factors/biosynthesis , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/immunology , Graft vs Host Disease/immunology , Humans , Interleukin-2/immunology , Lymphocyte Activation/immunology , Mice , Mice, SCID , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes, Regulatory/cytologyABSTRACT
In the present study, we examined the inhibitory effects of the beta2-adrenoceptor agonists isoproterenol, salbutamol, fenoterol, and clenbuterol, on the release of chemical mediators from cultured human mast cells after prolonged treatment with the agonists. Although preincubation of sensitized mast cells for 10 min with beta2-adrenoceptor agonists potently inhibited mediator release, prolongation of the preincubation period up to 240 min attenuated the inhibition. The attenuation of histamine release inhibition was potent when compared with that of prostaglandin D2 (PGD2) and cysteinyl leukotriene (LT) release inhibition. In contrast, forskolin inhibited mediator release and the inhibition increased gradually in proportion to the preincubation period. The reduced inhibition by the beta2-adrenoceptor agonists was compensated for by simultaneous treatment with cholera toxin. The beta2-adrenoceptor agonists elevated intracellular cAMP levels after 10-min incubation and the elevated levels were almost comparable to those after 240-min incubation. Forskolin elevated the intracellular cAMP levels more potently after incubation for 240 min than after 10 min. When mast cells were incubated for 3 d with the beta2-adrenoceptor agonists, similar attenuation of mediator release inhibition was observed. Elevation of intracellular cAMP levels was also attenuated, although beta2-adrenoceptor mRNA expression was potentiated. The present results collectively indicate that the attenuation of mediator release inhibition by beta2-adrenoceptor agonists under the present experimental conditions involves uncoupling between beta2-adrenoceptors and Gs proteins. Furthermore, the beta2-adrenoceptor desensitization causes differential attenuating effects on the inhibition of histamine, PGD2, and LT release, suggesting that downstream events involved in each inhibitory pathway have different sensitivity to receptor desensitization.
Subject(s)
Adrenergic beta-2 Receptor Agonists , Adrenergic beta-Agonists/pharmacology , Immunoglobulin E/metabolism , Mast Cells/drug effects , Actins/biosynthesis , Actins/genetics , Cells, Cultured , Colforsin/pharmacology , Cyclic AMP/agonists , Cyclic AMP/biosynthesis , Histamine Release/drug effects , Humans , Leukotriene D4/antagonists & inhibitors , Mast Cells/metabolism , Prostaglandin D2/antagonists & inhibitors , RNA, Messenger/biosynthesis , Receptors, Adrenergic, beta-2/biosynthesis , Receptors, Adrenergic, beta-2/genetics , Reverse Transcriptase Polymerase Chain Reaction , Time FactorsABSTRACT
NKT cells represent a unique subset of immunoregulatory T cells that recognize glycolipid Ags presented by the MHC class I-like molecule CD1d. Because of their immunoregulatory properties, NKT cells are attractive targets for the development of immunotherapies. The prototypical NKT cell ligand alpha-galactosylceramide (alpha-GalCer), originally isolated from a marine sponge, has potent immunomodulatory activities in mice, demonstrating therapeutic efficacy against metastatic tumors, infections, and autoimmune diseases, but also has a number of adverse side effects. In vivo administration of alpha-GalCer to mice results in the rapid activation of NKT cells, which is characterized by cytokine secretion, surface receptor down-regulation, expansion, and secondary activation of a variety of innate and adaptive immune system cells. In this study, we have evaluated the in vivo immune response of mice to a set of structural analogues of alpha-GalCer. Our results show that, contrary to current thinking, beta-anomeric GalCer can induce CD1d-dependent biological activities in mice, albeit at lower potency than alpha-anomeric GalCer. In addition, we show that the response of NKT cells to distinct GalCer differs not only quantitatively, but also qualitatively. These findings indicate that NKT cells can fine-tune their immune responses to distinct glycolipid Ags in vivo, a property that may be exploited for the development of effective and safe NKT cell-based immunotherapies.