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T Takai

Publications and source records attributed to T Takai.

At least 19 recordsLinked to original sources
PubMed2001

Drastic up-regulation of Fcepsilonri on mast cells is induced by IgE binding through stabilization and accumulation of Fcepsilonri on the cell surface.

It has been shown that IgE binding to FcepsilonRI on mast cells results in increased FcepsilonRI expression, which in turn enhances IgE-dependent chemical mediator release from mast cells. Therefore, prevention of the IgE-mediated FcepsilonRI up-regulation would be a promising strategy for management of allergic disorders. However, the mechanism of IgE-mediated FcepsilonRI up-regulation has not been fully elucidated. In this study, we analyzed kinetics of FcepsilonRI on peritoneal mast cells and bone marrow-derived mast cells. In the presence of brefeldin A, which prevented transport of new FcepsilonRI molecules to the cell surface, levels of IgE-free FcepsilonRI on mast cells decreased drastically during culture, whereas those of IgE-bound FcepsilonRI were stable. In contrast, levels of FcgammaRIII on the same cells were stable even in the absence of its ligand, indicating that FcepsilonRI alpha-chain, but not beta- and gamma-chains, was responsible for the instability of IgE-free FcepsilonRI. As far as we analyzed, there was no evidence to support the idea that IgE binding to FcepsilonRI facilitated synthesis and/or transport of FcepsilonRI to the cell surface. Therefore, the stabilization and accumulation of FcepsilonRI on the cell surface through IgE binding appears to be the major mechanism of IgE-mediated FcepsilonRI up-regulation.

Animals↗
PubMed2001

A critical role of Fc receptor-mediated antibody-dependent phagocytosis in the host resistance to blood-stage Plasmodium berghei XAT infection.

Plasmodium berghei XAT is an irradiation-induced attenuated variant derived from the lethal strain P. berghei NK65, and its blood-stage parasites are spontaneously cleared in immune competent mice. In the present study, we studied the mechanism of host resistance to blood-stage malaria infection using P. berghei XAT. Infection enhanced Ab-dependent phagocytosis of PRBC by splenic macrophages in wild-type C57BL/6 mice. In contrast, FcR gamma-chain knockout (FcRgamma(-/-)) mice, which lack the ability to mediate Ab-dependent phagocytosis and Ab-dependent cell-mediated cytotoxicity through FcgammaRI, FcgammaRII, and FcgammaRIII, could not induce Ab-dependent phagocytic activity. These FcRgamma(-/-) mice showed increased susceptibility to the P. berghei XAT infection, with eventually fatal results, although they produced comparable amounts of IFN-gamma by spleen cells and anti-XAT Abs in serum. In addition, passive transfer of anti-XAT IgG obtained from wild-type mice that had recovered from infection into FcRgamma(-/-) mice could not suppress the increase in parasitemia, and almost all of these mice died after marked parasitemia. In contrast, passive transfer of anti-XAT IgG into control wild-type mice inhibited the increase in parasitemia. IFN-gamma(-/-) mice, which were highly susceptible to the P. berghei XAT infection, failed to induce Ab-dependent phagocytic activity and also showed reduced production of serum anti-XAT IgG2a isotype compared with control wild-type mice. These results suggest that FcR-mediated Ab-dependent phagocytosis, which is located downstream of IFN-gamma production, is important as an effector mechanism to eliminate PRBC in blood-stage P. berghei XAT infection.

Animals↗
PubMed2001

Lyn is essential for fcgamma receptor III-mediated systemic anaphylaxis but not for the Arthus reaction.

The Src family kinase Lyn initiates intracellular signal transduction by associating with a variety of immune receptors such as antigen receptor on B cells and high-affinity Fc receptor (FcR) for immunoglobulin Ig(E) (FcepsilonRI) on mast cells. Involvement of Lyn in the IgE-mediated immediate-type hypersensitivity is well documented, but the physiological significance of Lyn in IgG-dependent, type III low-affinity FcR for IgG (FcgammaRIII)-mediated responses is largely unknown. In this study, we generated a double-mutant mouse strain deficient in both type II FcR for IgG (FcgammaRIIB) and Lyn to exclude any involvement of inhibitory signaling by FcgammaRIIB, which otherwise downregulates FcgammaRIII-mediated cellular responses. FcgammaRIIB-deficient but Lyn-sufficient mice served as controls. The Lyn deficiency attenuated IgG-mediated systemic anaphylaxis in vivo, and significantly reduced calcium mobilization and degranulation responses of bone marrow-derived mast cells (BMMCs) in vitro. However, we found that either interleukin 4 or tumor necrosis factor alpha release by BMMCs was comparable to that from Lyn-deficient and control mice, and the reverse-passive Arthus reaction was equally induced in both mutant mice, indicating that Lyn is not involved in the onset of the IgG-mediated, FcgammaRIII-dependent late phase responses of mast cells. These findings provide us with insight into distinct signaling mechanisms in mast cells underlying the development of diverse pathologies as well as a therapeutic potential for selective treatment of allergic disorders.

Anaphylaxis↗
PubMed2001

A comparative vibrational and NMR study of cis-cinnamic acid polymorphs and trans-cinnamic acid.

The IR and Raman spectra of the two polymorphic forms (58 degree- and 68 degree-forms) of cis-cinnamic acid were measured, and the spectral differences discussed on the basis of the crystal structures of the two forms. The IR bands related to the COOH group differ in the frequencies and band shape, reflecting differences in the hydrogen bonding between the two modifications. These spectra were compared with those of trans-cinnamic acid. The IR, Raman, and NMR spectra of the isotopic compounds, including the deuterated and 13C analogs of the cis and trans acids, were also recorded in the solid state and in solution to confirm the spectral assignments.

Cinnamates↗
PubMed2001

Functional association of CD9 with the Fc gamma receptors in macrophages.

CD9, a member of the tetraspan family of proteins, is highly expressed on macrophages. Although a clear function for the molecule has yet to be described, we have found that the anti-CD9 mAb activates mouse macrophages. The rat anti-CD9 mAb, KMC8.8, but not the F(ab')(2), induced tyrosine phosphorylation of proteins including syk and cbl and induced cell aggregation in the mouse macrophage cell line, J774, suggesting that co-cross-linking of CD9 and Fc gamma R was required for the signal. Co-cross-linking of CD9-Fc gamma R with KMC8.8 on macrophages from three different FcR-deficient mice, FcR gamma-chain(-/-), Fc gamma RIIB(-/-), and Fc gamma RIII(-/-), revealed that Fc gamma RIII is specific and crucial for syk phosphorylation. Although both KMC8.8 and the anti-Fc gamma RIIB/III mAb, 2.4G2, evoked similar phosphorylation patterns, only KMC8.8 induced cell aggregation. Additionally, KMC8.8 treatment led to reduce levels of TNF-alpha production and p42/44 extracellular signal-related kinase phosphorylation relative to 2.4G2 stimulation. Immunofluorescence staining showed that co-cross-linking of CD9-Fc gamma R with KMC8.8 induced filopodium extension before cell aggregation, which was followed by simultaneous colocalization of CD9, Fc gamma RIIB/III, Mac-1, ICAM-1, and F-actin at the cell-cell adhesion site. Moreover, KMC8.8 treatment of Fc gamma R-deficient macrophages revealed that the colocalization of CD9, Fc gamma RIII, Mac-1, and F-actin requires co-cross-linking of CD9-Fc gamma RIII, whereas co-cross-linking of CD9-Fc gamma RIIB induced the colocalization of only CD9 and Fc gamma RIIB. Our results demonstrate that co-cross-linking of CD9 and Fc gamma Rs activates macrophages; therefore, CD9 may collaborate with FcRs functioning in infection and inflammation on macrophages.

Actins↗
PubMed2001

Activating and inhibitory nature of the murine paired immunoglobulin-like receptor family.

Clones for murine paired immunoglobulin-like receptors (PIR) were first isolated as those coding for type I transmembrane glycoproteins with six immunoglobulin-like domains homologous to human Fc alphaR, bovine Fc gamma2R, and other related receptors. However, they turned out to bind neither IgA nor other immunoglobulins in the case of the ectopic expression on COS-1 fibroblastic cells. PIR-A and B are expressed on a wide variety of cells in the murine immune system, such as in B cells, mast cells, macrophages, and dendritic cells, mostly in a pairwise fashion. PIR-A requires homodimeric Fc receptor common gamma chain, which harbors an immunoreceptor tyrosine-based activation motif, for its efficient cell surface expression and for the delivery of activation signaling. In contrast, PIR-B contains immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic portion and inhibits receptor-mediated activation signaling in vitro upon engagement with other activating-type receptors such as the antigen receptor on B cells and the high affinity Fc receptor for IgE on mast cells. ITIMs of PIR-B on macrophages and B cells have been shown to be constitutively phosphorylated in their tyrosine residues. Although the ligand for PIR still remains unknown, the transgenics and the gene-targeted mice will provide us with valuable information on their physiological roles in the immune regulation.

Animals↗
PubMed2001

Biologically active recombinant forms of a major house dust mite group 1 allergen Der f 1 with full activities of both cysteine protease and IgE binding.

BACKGROUND: Group 1 allergens from mite faeces, Der f 1 and Der p 1, are the most significant in-door allergens. Therefore, they are the most important component in the standardization of house dust mite extract for diagnosis and allergen-specific immunotherapy (AIT). Although their cDNAs have been cloned, efforts to prepare biologically active recombinant forms in expression systems using bacteria or yeast have failed. OBJECTIVE: Our purpose is to establish an efficient system to prepare recombinant Der f 1(rDer f 1), identical in quality to native Der f 1. METHODS: The preproforms of Der f 1 and a mutant N53Q, whose consensus motif for N-glycosylation was disrupted, were expressed in yeast Pichia pastoris. Cysteine protease activity and IgE reactivity were analysed using synthetic substrates and by RAST-EIA, respectively. RESULTS: The proforms of the two rDer f 1 molecules were efficiently secreted into culture medium. Their prosequences were removed autocatalytically by dialysis against acidic buffer. Although the wild-type rDer f 1 was more highly glycosylated than native Der f 1, N53Q had almost the same apparent molecular weight as native Der f 1 on SDS-PAGE. Both the protease and IgE binding activities of the mature rDer f 1 molecules were the same as those of native Der f 1, whereas the proforms had no or markedly reduced activities. CONCLUSION: The efficient system to prepare active rDer f 1s established in this study is useful for diagnosis and standardized AIT for house dust mite allergy. Furthermore, the system would be a tool for analysis of IgE epitopes, determination of tertiary structure, allergen engineering for safer and more effective AIT, resolving the relation between the enzymatic activity and pathogenesis, and the development of therapeutic inhibitors.

Animals↗
PubMed2001

Expression of humanized Fab fragments that recognize the IgE-binding domain of human Fc(epsilon)RIalpha in COS and CHO cells.

Interfering with the binding of IgE to high-affinity IgE receptor alpha chain (Fc(epsilon)RIalpha) is a straightforward strategy for the specific prevention of the IgE-mediated allergic reaction specifically. A Fab fragment (Fab) of a humanized antibody against the membrane proximal IgE-binding domain of human Fc(epsilon)RIalpha inhibits the release of histamine from human basophils. We established an efficient expression system in which to produce directly the humanized anti-human Fc(epsilon)RIalpha Fabs without papain-digestion of the whole antibody. Four Fabs with different C-termini of CH1 were expressed directly in COS-7 cells transfected with expression vectors with or without the Fc gene downstream of a stop codon inserted within the hinge gene. The secretion of Fabs when transfected without the Fc gene was remarkably enhanced compared to that when transfected with the Fc gene. The ability of Fabs to inhibit IgE-Fc(epsilon)RIalpha binding when transfected without the Fc gene was equivalent to that of purified Fab prepared by papain-digestion of the whole antibody. No significant differences among the four Fabs were observed in secretion or activity. Clones of CHO-transfectant cells that secreted the Fabs constitutively were acclimatized to a serum-free medium. Analysis of the binding interface between the Fab and human Fc(epsilon)RIalpha will provide useful information for the design of therapeutic reagents for allergy and asthma.

Animals↗
PubMed2001

Effects of site-directed mutagenesis in the cysteine residues and the N-glycosylation motif in recombinant Der f 1 on secretion and protease activity.

BACKGROUND: The group 1 allergens from mite feces, which belong to the papain-like cysteine protease family, are the most significant in-door allergens. In this study, we analyzed the contribution of the cysteine residues and N-glycosylation in Der f 1, the group 1 allergen from Dermatophagoides farinae, to secretion and maturation by using systems for expression of recombinant Der f 1 (rDer f 1). METHODS: The rDer f 1 and its mutants were expressed in yeast Pichia pastoris and insect SF9 cells. Secretion of their proforms was checked by SDS-PAGE or immunoblotting. Protease activities of the secreted proform of a mutant and the mature form were compared with that of native Der f 1. RESULTS: The proform of a mutant Der f 1, pro-N53Q, whose consensus motif for N-glycosylation was disrupted, was not secreted in insect SF9 cells although secreted in P. pastoris. Indirect evidence was obtained to support the disulfide bond formation between Cys4 and Cys118, which were not conserved in papain. A mutant for Cys35 in the catalytic site of the cysteine protease, pro-C35S/N53Q, was secreted, but the other mutants for cysteines concerning intramolecular disulfide bonds were not secreted in P. pastoris. The prosequence of pro-C35S/N53Q was removed by an in vitro activation process. The mature C35S/N53Q showed low protease activity. CONCLUSION: N-glycosylation is essential for secretion in insect SF9 cells but not in P. pastoris. Disulfide bonds are essential for secretion in P. pastoris. A mutation in the catalytic site, C35S, is not completely critical to removal of the prosequence and protease activity. The findings are useful for future design of recombinant products for application in immunotherapy.

Amino Acid Motifs↗
PubMed2001

Production of humanized antibody against human high-affinity IgE receptor in a serum-free culture of CHO cells, and purification of the Fab fragments.

We describe the preparation of Fab fragments of a humanized anti-human high-affinity IgE receptor (Fc epsilonRIalpha) antibody potentially useful for treatment of IgE-mediated allergic diseases. IgE-binding capacities of sixteen combinations of light and heavy chains of four recombinant anti-Fc epsilonRIalpha antibodies, chimeric CRA2, humanized CRA2, chimeric CRA4, and humanized CRA4, were compared. A combination in which both chains were of humanized CRA2 had the highest activity. Stable transfectant clones of four kinds of host cells expressing recombinant antibodies were established. CHO-K1 cells were the most productive. Serum-free media suitable for culture of the stable CHO-transfectant clones were screened. The concentration of the humanized CRA2, which the most productive clone secreted into the chosen serum-free medium, was approximately 100 microg/ml. A procedure for the purification of the antibody, papain-digestion, and purification of Fab fragments was established. The highly purified humanized Fab fragments are suitable for use to examine their in vivo activity and immunogenicity in primates.

Animals↗
PubMed2001

Effects of double mutation at two distant IgE-binding sites in the three-dimensional structure of the major house dust mite allergen Der f 2 on IgE-binding and histamine-releasing activity.

Recently, we reported that introduction of mutations that induced conformational changes of the major mite allergen Der f 2 was an efficient strategy to reduce the allergenicity for safer allergen-specific immunotherapy. In this study, we evaluated another strategy, disruption of two independent IgE epitopes without inducing conformational change. We analyzed allergenicities of the wild-type Der f 2, two single mutants with a mutation at either of the two IgE-binding sites (K15A and K77A), and a double mutant with mutations at both of the sites (K15/77A). Purified recombinant forms of Der f 2 expressed in Escherichia coli had correct disulfide bonds, equivalent apparent molecular masses of approximately 15 kDa, and similar secondary structures. The mutants of Der f 2 had less IgE reactivities than the wild-type Der f 2 and reduced inhibitory activities for IgE-binding to the wild-type Der f 2. However, the mutations did not significantly reduce histamine-releasing activity.

Allergens↗
PubMed2001

Cloning and expression of cDNA encoding the complete prepro-form of an isoform of Der f 1, the major group 1 allergen from house dust mite Dermatophagoides farinae.

cDNA clones encoding a major house dust mite allergen, Der f 1, were isolated from a Dermatophagoides farinae cDNA library by plaque immunoscreening using rabbit anti-Der f 1 serum. The sequences cover the complete open reading frame encoding the prepro-form. The sequence is different from previously reported cDNA of Der f 1 in six bases and the encoded amino acid sequence is different in two residues. Pro-forms of Der f 1 and its mutant, in which the N-glycosylation motif was disrupted, expressed in Pichia pastoris were converted to the mature forms by an in vitro activation process and they showed significant IgE-binding. The biologically active rDer f 1 molecules would be useful for diagnostic testing and allergen-specific immunotherapy. In contrast, Der f 1 directly expressed in Escherichia coli without the prosequence had very low IgE binding. The hypoallergenic Der f 1 polypeptide could be useful for safer and more effective immunotherapy.

Allergens↗
PubMed2001

Reactivities of mutants of a major house dust mite allergen Der f 2 to mouse anti-Der f 2 monoclonal antibodies analyzed by immunoblotting.

A total of sixteen recombinant variants of a major house dust mite allergen Der f 2, the wild-type Der f 2, six cysteine mutants, six proline mutants, and three lysine mutants, were expressed in Escherichia coli. The cells were solubilized and run on SDS-PAGE under reducing conditions. Epitopes for five mouse anti-Der f 2 monoclonal antibodies, 1B2, 7C10, 13A4, 15E11, and 18G8, to the recombinant Der f 2 variants were characterized by immunoblot analysis.

Allergens↗
PubMed2001

Direct expression of the extracellular portion of human FcepsilonRIalpha chain as inclusion bodies in Escherichia coli.

The extracellular portion of the alpha chain of the human high-affinity IgE receptor (FcepsilonRIalpha) was expressed as inclusion bodies in Escherichia coli. In immunoblot analysis, two bands were reactive to human IgE and mouse anti-human FcepsilonRIalpha monoclonal antibodies. N-terminal sequencing showed that the two bands were equivalent to the soluble FcepsilonRIalpha with a methionine residue at the N-terminus (Met-1-172) and 23-172, in which the N-terminal 22 residues of the soluble FcepsilonRIalpha have been removed, possibly by degradation in E. coli cells. IgE-binding to CHO cells expressing FcepsilonRI was inhibited by the addition of the recombinant products prepared by the refolding procedure from inclusion bodies. The system for the expression of soluble human FcepsilonRIalpha in E. coli presented in this study and its further improvement would be useful for the production of the protein as a potent therapeutic and for analysis of the IgE-FcepsilonRIalpha interaction.

Animals↗
PubMed2000

Unlocking the allergenic structure of the major house dust mite allergen der f 2 by elimination of key intramolecular interactions.

We report on the structural background of the remarkable reduction of allergenicity in engineering of the major house dust mite allergen Der f 2. Disruption of intramolecular disulfide bonds in Der f 2 caused extensive conformational change that was monitored by circular dichroism and gel-filtration analysis. The degree of conformational change correlated well with the degree of reductions in the capacity to bind IgE and to induce histamine release from basophils in mite-allergic patients. Loosening the rigid tertiary structure by elimination of key intramolecular interactions is an effective strategy to reduce the number of high affinity IgE epitopes of allergen vaccine.

Animals↗
PubMed2000

Stimulatory function of gp49A, a murine Ig-like receptor, in rat basophilic leukemia cells.

Murine gp49, a 49-kDa type I transmembrane glycoprotein, is a member of the Ig-like receptors expressed on the surface of cells involved in natural immunity such as mast cells, NK cells, and macrophages. The two major subtypes, gp49A and gp49B, are encoded by two different genes adjacent to each other. gp49B contains an immunoreceptor tyrosine-based inhibitory motif in its cytoplasmic region and is known to function as an inhibitory molecule. In contrast, gp49A does not harbor any specific motif for signal transduction, nor has its physiological role been determined. Here we report on the stimulatory nature of gp49A by analyzing biochemical characteristics of chimeric molecules consisting of an ectodomain of Fc receptor and a C-terminal half of gp49A, namely the pretransmembrane, transmembrane, and cytoplasmic portions, expressed on the rat basophilic leukemia mast cell line. Cross-linking of the chimeric receptors evoked cytoplasmic calcium mobilization, PGD(2) release, and transcription of IL-3 and IL-4 genes, but did not elicit degranulation of the cells. The chimeric molecule could be expressed as a singlet and a homodimeric form on the cell surface. A pretransmembrane cysteine residue of gp49A was necessary for dimer formation. Dimerization was be necessary for their incorporation into glycolipid-enriched membrane fraction (GEM) upon cross-linking stimuli. The calcium mobilization response was inhibited by treatment of cells with methyl-beta-cyclodextrin, an inhibitor of GEM formation. Together with these results, it was strongly suggested that gp49A could be expressed as a homodimer and elicit activation signals that lead to calcium mobilization, eicosanoid production, and cytokine gene transcription through its incorporation into GEM.

Animals↗
PubMed2000

Fcgamma receptor IIB-deficient mice develop Goodpasture's syndrome upon immunization with type IV collagen: a novel murine model for autoimmune glomerular basement membrane disease.

The combination of hemorrhagic pneumonitis and rapidly progressive glomerulonephritis is a characteristic feature of Goodpasture's syndrome (GPS), an autoimmune disease resulting from the interaction of pathogenic anti-collagen type IV (C-IV) antibodies with alveolar and glomerular basement membranes. Lack of a suitable animal model for this fatal disease has hampered both a basic understanding of its etiology and the development of therapeutic strategies. We now report a novel model for GPS using mice deficient in a central regulatory receptor for immunoglobulin (Ig)G antibody expression and function, the type IIB Fc receptor for IgG (FcgammaRIIB). Mutant mice immunized with bovine C-IV reproducibly develop massive pulmonary hemorrhage with neutrophil and macrophage infiltration and crescentic glomerulonephritis. The distinctive linear, ribbon-like deposition of IgG immune complex seen in GPS was observed along the glomerular and tubulointerstitial membranes of diseased animals. These results highlight the role of FcgammaRIIB in maintaining tolerance and suggest that it may play a role in the pathogenesis of human GPS.

Animals↗