[Immunoregulatory functions of Fc receptors].
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Animals↗
Biomedical subjects
T Takai
Publications and source records attributed to T Takai.
Reexpression of RAG-1 and RAG-2 genes in activated mature mouse B cells.
Recombination activating genes (RAG-1 and RAG-2), involved in V(D)J rearrangement of immunoglobulin genes, have been thought to be expressed only in immature stages of B-cell development. However, RAG-1 and RAG-2 transcripts were found to be reexpressed in mature mouse B cells after culture with interleukin-4 in association with several different co-stimuli. Reexpression was also detected in draining lymph nodes from immunized mice. RAG-1 and RAG-2 proteins could be detected by immunofluorescence microscopy in the nuclei of B cells cultured in vitro and in the germinal centers of draining lymph nodes. These findings suggest that RAG gene products play a heretofore unsuspected role in mature B cells.
Animals↗
Requirements of a costimulus for IL-4-induced IgE class switching in murine B cells activated via antigen receptors: effectiveness of 8-mercaptoguanosine.
In the present paper, we investigated the requirements for the induction of IgE responses in murine B cells stimulated via surface Ig receptors by trinitrophenyl (TNP)-LPS or F(ab')2 of anti-mouse IgM (anti-mu) Abs. When murine B cells were cultured with 0.2 microgram/ml TNP-LPS, the secretion of anti-TNP IgM, but not that of IgG1 or IgE, was induced. It should be noted that IL-4, alone or in combination with IL-2 or IL-5, was not sufficient for inducing IgE class switching in these B cells, in contrast to the cells activated polyclonally with 20 micrograms/ml LPS, suggesting that an additional signal is required under the former conditions. After extensive screening of costimuli for inducing IgE in cooperation with IL-4, we found that 8-mercaptoguanosine (8-SGuo), a potent B cell activator, was effective. The addition of 8-SGuo to TNP-LPS-stimulated B cell cultures resulted in the secretion of both anti-TNP IgM and IgG1, but not anti-TNP IgE. Anti-TNP IgE formation was induced only when 8-SGuo was added in combination with IL-4, whereas 8-SGuo plus IL-4 failed to induce IgE secretion in the absence of TNP-LPS. Similar requirements for 8-SGuo and IL-4 for IgE formation were observed when B cells were stimulated with anti-mu. It was revealed that induction of the IgE responses was accompanied by increased frequency of surface IgE+ B cells and the expression of germline and productive epsilon transcripts. In B cells stimulated with anti-mu plus IL-4, 8-SGuo was shown to enhance the expression of the B cell-specific activator protein gene that is thought to be required in IL-4-dependent germline epsilon transcription.
Adjuvants, Immunologic↗
Augmented humoral and anaphylactic responses in Fc gamma RII-deficient mice.
Despite its widespread distribution on both lymphoid and myeloid cells, the biological role of the low-affinity immunoglobulin-G receptor, Fc gamma RII, is not fully understood. Defects in this receptor or its signalling pathway in B cells result in perturbations in immune-complex-mediated feedback inhibition of antibody production. We now report that Fc gamma RII-deficient animals display elevated immunoglobulin levels in response to both thymus-dependent and thymus-independent antigens. Additionally, the effector arm of the allergic response is perturbed in these mice. Mast cells from Fc gamma RII-/- are highly sensitive to IgG-triggered degranulation, in contrast to their wild-type counterparts. Fc gamma RII-deficient mice demonstrate an enhanced passive cutaneous analphylaxis reaction, the result of a decreased threshold for mast-cell activation by Fc gamma RIII cross-linking. These results demonstrate that Fc gamma RII acts as a general negative regulator of immune-complex-triggered activation in vivo for both the afferent and efferent limbs of the immune response. Exploiting this property offers new therapeutic opportunities for the treatment of allergic and autoimmune disorders.
Animals↗
Radioimmunoassay for imidapril, a new angiotensin-converting enzyme inhibitor, and imidaprilat, its active metabolite, in human plasma and urine.
A radioimmunoassay (RIA) was investigated for the determination of imidapril and its active metabolite, imidaprilat, in human plasma and urine. Imidapril is a new angiotensin-converting enzyme inhibitor and an oral prodrug of imidaprilat. Imidapril was determined after conversion to imidaprilat with esterase. Antiserum was raised in rabbits against the p-amino derivative of imidaprilat conjugated to bovine serum albumin. Radioligand was prepared by iodination (125I) of the p-hydroxybenzoylamino derivative of imidaprilat. Cross-reactivities of anti-imidaprilat antiserum for imidapril, its metabolites and several cardiovascular drugs were low. The calibration range was 0.1-100 ng ml-1 using a 100 microliters of human plasma of urine. Intra- and inter-day variations of imidaprilat assay in plasma were 2.0-7.9 and 4.1-6.2%, respectively, and intra- and inter-day variations of imidapril assay in plasma were 5.4-10.7 and 7.9-18.1%, respectively. The variations of the assay in urine were a little smaller than those in plasma. The recovery of imidaprilat and imidapril spiked in plasma or urine samples was approximately 100%. A good correlation between RIA and high-performance liquid chromatograpy was observed for both plasma and urine samples. Furthermore, this method was applied to the determination of imidaprilat and imidapril in human plasma and urine samples, for the evaluation of the pharmacokinetics of imidapril in humans. From the results, it was demonstrated that the developed RIA was useful for the determination of imidaprilat and imidapril in human plasma and urine, and was applicable to pharmacokinetic studies in humans.
Angiotensin-Converting Enzyme Inhibitors↗
Suppression of in vitro cellular immune response by nitrogen-containing terpene alcohol derivatives.
A series of nitrogen-containing terpene alcohol derivatives were tested for their immunosuppressive effects in vitro, and KYKC-407 (erythro-1-(1-imidazolyl)-3,7,11-trimethyl-dodecane-2(S),3(R)-diol) was found to be the most effective in suppressing the induction of cytotoxic T cell (CTL) activity. CTL induction in the coculture of C3H spleen cells with mitomycin C-treated BALB/c spleen cells was suppressed by more than 90% when 4 microM KYKC-407 was added to the culture. Under these conditions, lymphocyte proliferation was also suppressed to a similar extent by this compound. Flow cytometric analysis revealed that KYKC-407 strongly inhibited the proliferation of CD8+ T cell subset, but showed less suppressive effects on CD4+ T cells. In contrast to cyclosporin A, KYKC-407 at 1-4 microM did not inhibit the proliferative response to concanavalin A. Further, KYKC-407 suppressed CTL induction even in the presence of exogenous IL-2, thus suggesting that the compound does not exert its effects through inhibiting IL-2 production.
Animals↗
Multiple loss of effector cell functions in FcR gamma-deficient mice.
Immunoglobulin Fc receptor (FcR) gamma subunit is a component of low affinity receptor for IgG, Fc gamma RIII, as well as high affinity receptor for IgE, Fc epsilon RI. This subunit is required for efficient surface expression of these FcRs on various cells in immune system. The FcR gamma-deficient mice, generated by gene targeting in embryonic stem cells, exhibit multiple defects in FcR-mediated effector cell responses, including absence of phagocytic activity against opsonized red blood cells by activated macrophages, loss of antibody-dependent cell-mediated cytotoxicity manifested by IL-2-induced splenic NK cells, and unresponsiveness of mast cells to crosslinking of IgE on these cells. These results demonstrate an indispensable role of FcR gamma for functional expression of FcRs, and clearly indicate the importance of Fc gamma RIII as well as Fc epsilon RI for these effector functions. Since FcR gamma-deficient mice is unable to mount the type II and type III hypersensitivity reactions, it is suggested that FcRs play pivotal roles in initiating these reaction cascades. The mutant mice should prove to be useful in evaluating FcRs in various humoral and cellular immune responses, and in developing new strategies for treatment of immunodeficiency as well as autoimmune disorders.
Animals↗
[Infectious diseases of the liver due to protozoa].
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Animals↗
Molecular cloning of MSSP-2, a c-myc gene single-strand binding protein: characterization of binding specificity and DNA replication activity.
We have previously reported the human cDNA encoding MSSP-1, a sequence-specific double- and single-stranded DNA binding protein [Negishi, Nishita, Saëgusa, Kakizaki, Galli, Kihara, Tamai, Miyajima, Iguchi-Ariga and Ariga (1994) Oncogene, 9, 1133-1143]. MSSP-1 binds to a DNA replication origin/transcriptional enhancer of the human c-myc gene and has turned out to be identical with Scr2, a human protein which complements the defect of cdc2 kinase in S.pombe [Kataoka and Nojima (1994) Nucleic Acid Res., 22, 2687-2693]. We have cloned the cDNA for MSSP-2, another member of the MSSP family of proteins. The MSSP-2 cDNA shares highly homologous sequences with MSSP-1 cDNA, except for the insertion of 48 bp coding 16 amino acids near the C-terminus. Like MSSP-1, MSSP-2 has RNP-1 consensus sequences. The results of the experiments using bacterially expressed MSSP-2, and its deletion mutants, as histidine fusion proteins suggested that the binding specificity of MSSP-2 to double- and single-stranded DNA is the same as that of MSSP-1, and that the RNP consensus sequences are required for the DNA binding of the protein. MSSP-2 stimulated the DNA replication of an SV40-derived plasmid containing the binding sequence for MSSP-1 or -2. MSSP-2 is hence suggested to play an important role in regulation of DNA replication.
Base Sequence↗
FcR gamma chain deletion results in pleiotrophic effector cell defects.
The gamma subunit of immunoglobulin Fc receptors is an essential component of the high-affinity receptor for IgG (Fc gamma RIII) and is associated with the high-affinity receptor for IgG (Fc gamma RI) and the T cell receptor-CD3 complex. It is required for both receptor assembly and signal transduction. Targeted disruption of this subunit results in immunocompromised mice. Activated macrophages from gamma chain-deficient mice unexpectedly lack the ability to phagocytose antibody-coated particles, despite normal binding. Defects in NK cell-mediated antibody-dependent cytotoxicity and mast cell-mediated allergic responses are evident in these animals, establishing the indispensable role of FcRs in these responses. However, loss of gamma chain does not appear to perturb T cell development, since both thymic and peripheral T cell populations appear normal. These mice thus represent an important tool for evaluating the role of these receptors in humoral and cellular immune responses.
Animals↗
Predominant suppression of anti-TNP IgE response in mice by monoclonal anti-TNP IgG1 antibody: characterization of its mode of action by in vitro and in vivo studies.
It was found that an antigen-specific IgE response both in vitro and in vivo was strongly suppressed in the presence of IgG1 monoclonal antibody (mAb) against the antigen. Anti-trinitrophenyl (TNP) IgE response was elicited by the co-culture of C3H B-cells and a conalbumin (CA)-specific helper T-cell clone, D10.G4.1, in the presence of 0.1 microgram/ml TNP-CA. Addition of anti-TNP IgG1 monoclonal antibody (mAb) at 1 microgram/ml to the culture resulted in a marked (> 90%) suppression of anti-TNP IgE formation, while anti-TNP IgG1 and IgM responses were affected to a lesser extent (50-60% suppression). Similar observations were made in in vivo experiments. When 100-200 micrograms of anti-TNP IgG1 mAb was injected i.p. into BDF1 mice prior to immunization with TNP-CA, the anti-hapten (TNP) IgE response as well as the IgE response to the carrier (CA) was suppressed by 80-90%, while anti-TNP IgM production was inhibited by less than 50%. Injection of anti-TNP IgM or IgA mAb showed only marginal effects on anti-TNP IgE production. Spleen cells from anti-TNP IgG1 mAb-treated mice cultured in vitro secreted much lower levels of anti-TNP IgE spontaneously than those from untreated mice. In in vitro and in vivo experiments using the F(ab')2 of anti-TNP IgG1 mAb, an IgG1 mAb with an irrelevant specificity and mAb directed to Fc gamma RII, it was shown that the binding of the IgG1 mAb with the antigen and the interaction of its Fc portion with Fc gamma RII are required for the suppressive effects to be exerted.
Animals↗
Nature of protein adsorption in artificial tear solution on plasma-irradiated polymethylmethacrylate surface.
To evaluate the effect of plasma treatment on protein adsorption onto a polymer surface, we carried out Ar plasma irradiation on polymethylmethacrylate (PMMA) plate and examined the adsorption behavior of protein after immersion of plasma treated PMMA plate in the artificial tear solution containing lysozyme, albumin and gamma-globulin. It was found that the total adsorption of protein in the artificial tear solution to PMMA is suppressed by the Ar plasma treatment. But the adsorption of each component protein differs according to the surface condition of polymers and obviously corresponds to the change with time in the surface wettability. It is concluded that introduction of a hydrophilic group into the PMMA surface by plasma treatment and coming off of it are associated with the adsorption behavior of proteins. Since the adsorption of protein to the plasma-treated PMMA surface changes over time, care should be exercised in determining it, when an attempt is made to clarify the effect of plasma irradiation on the adsorption to the plasma-irradiated polymer surface.
Adsorption↗
Phorbol ester-induced reversible inactivation of cytotoxic T cell function: correlation with down-regulation of protein kinase C activity.
When an H-2d-specific cytotoxic T lymphocytes (CTL) clone, FC1, was incubated in the presence of 10(-7) M phorbol myristate acetate (PMA) for 10-12 hr, the cytolytic activity of the CTL against H-2d target cells was abrogated, but was reversibly restored to the normal level after subsequent incubation of the cells in PMA-free medium for more than 10 hr. These effects of PMA have been reported (Russell, J.H.: J. Immunol. 133, 907-912 (1984)), but the mode of its action has not been fully investigated. Here, we analyzed the biochemical basis of the PMA-induced loss of cytolytic activity. Cycloheximide completely blocked the restoration of the PMA-suppressed cytolytic activity, suggesting that protein synthesis was required in this process. PMA-treatment did not affect the levels of CD3 and CD8 molecules expressed on the CTL, nor was the level of a CTL-specific serine esterase, BLT esterase, affected by this treatment. However, the target cell-induced release of BLT esterase from the CTL was suppressed if the cells were pretreated with PMA. PMA-treatment of the CTL led to the down-regulation of protein kinase C (PKC) activity by about 50%. On the other hand, staurosporin, an inhibitor of PKC, completely blocked the target cell lysis when added at 10(-6) M. These results suggest that the down-regulation of at least some isoform(s) of PKC is responsible for the PMA-induced loss of the cytotolytic activity of CTL.
Animals↗
Histopathological study of intrahepatic aberrant vessels in cases of noncirrhotic portal hypertension.
Aberrant vessels, which are defined as dilated blood vessels immediately adjacent to the peripheral portal tract, appear under conditions of extrahepatic portal obstruction and nodular regenerative hyperplasia as well as idiopathic portal hypertension. Our study was undertaken to compare their morphological aspects in these three disease cases. Aberrant vessels were found in 84% of cases of idiopathic portal hypertension, 67% of cases of extrahepatic portal obstruction infantile type, 78% of cases of extrahepatic portal obstruction adult type and 83% of cases of nodular regenerative hyperplasia. They were divided into three types: type I--no communication with the portal vein, the lumen of which is normally open; type II--communication with the portal vein; and type III--no communication with the portal vein, which is occluded. The most common types of aberrant vessel were type III in idiopathic portal hypertension (51%), type I in extrahepatic portal obstruction infantile type (46%), type II in extrahepatic portal obstruction adult type (43%) and type III in nodular regenerative hyperplasia (45%). Serial sections revealed transition between types I, II and III, at frequencies between types II and III, types I and II, and types I and III of 35.7%, 33.7% and 30.6%, respectively. Aberrant vessels demonstrated the same immunoreactivity as portal veins for collagen type IV, laminin, factor VIII and ulex europaeus agglutinin-I. They were concluded to arise from the vasa septalis or inlet venules, which would be used as intrahepatic shunts draining portal blood flow blocked by stenosed portal veins. Increased portal pressure would be expected to enhance development of aberrant vessels.(ABSTRACT TRUNCATED AT 250 WORDS)
Blood Vessels↗
Enzyme release assay of human NK cell activity using beta-galactosidase-expressing K562 target cell line.
In the present report, we established a K562 cell line useful for an enzyme release assay of human natural killer (NK) activity. Human myelogenous leukemia cell line, K562, was transfected with a plasmid carrying Escherichia coli beta-galactosidase (beta-gal) gene. A colony that permanently expresses the enzyme activity was isolated, and designated K562/Zneo. Incubation of K562/Zneo cells (1 x 10(4)) with nonadherent human peripheral blood lymphocytes (PBL) resulted in the release of beta-gal activity depending on the incubation time and the number of effector cells. Released beta-gal activity was assayed sensitively by using 4-methylumbelliferyl-beta-D-galactoside, a fluorescent substrate. The cytolytic activity of PBL was augmented significantly when the cells were preincubated with interleukin-2 for 20 h. This enzyme release assay showed a comparable sensitivity to that of 51Cr release assay. Thus, K562/Zneo cell line is thought to be useful for the nonradioactive assay of human NK and lymphokine-activated killer activities.
Cytotoxicity, Immunologic↗
Determination of three disulfide bonds in a major house dust mite allergen, Der f II.
Der f II is a major mite allergen consisting of 129 amino acid residues. Der f II contains six cysteine residues, suggesting the existence of three disulfide bonds which would stabilize this small protein. As the first step in revealing the relationship between the structure and the allergenic property of Der f II, the formation of disulfide bonds was examined. Der f II purified from Dermatophagoides farinae was treated with lysyl-endopeptidase or proline-specific endopeptidase, and the peptide fragments thus generated were separated by reverse phase high performance liquid chromatography. Determination of the amino acid sequence of each peptide collected in this way proved the existence of three disulfide bonds between Cys8 and Cys119, Cys21 and Cys27, and Cys73 and Cys78.
Allergens↗
Establishment of an enzyme release assay for cytotoxic T lymphocyte activity.
In the present report, we describe the establishment of a cell line that can be used as the target for measuring the activity of cytotoxic T lymphocytes (CTL) by an enzyme release assay. We transfected P3/NS1-Ag4-1 (NS-1), a myeloma cell line derived from BALB/c mice with Escherichia coli beta-galactosidase (beta-Gal) gene, and isolated a stable transformant designated as NS-1/Z that expressed a high level of the enzyme activity intracellularly. The effector cells showing cytotoxicity against NS-1/Z were induced when the spleen cells of AKR or C3H mice were cultured with mitomycin C-treated BALB/c spleen cells for 4 days. When 2 x 10(4) NS-1/Z cells were incubated with varying numbers of effector cells, beta-Gal activity was released from the target cells depending on the number of effector cells and the time of incubation for up to 8 h. A highly sensitive enzyme assay was performed by using a fluorescent substrate, 4-methylumbelliferyl-beta-D-galactoside. The cytotoxicity was specific for H-2 haplotype of the stimulator cells, and was abolished by treating the effector cells with anti-Lyt 2 plus complement. The sensitivity of the enzyme release assay was comparable to that of 51Cr release assay. These results indicate that NS-1/Z can be used as a target cell line for the non-radioactive measurement of CTL activity.
Animals↗