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Y Shinkai

Publications and source records attributed to Y Shinkai.

At least 55 records · Page 3Linked to original sources
PubMed1992

Expression of perforin in murine natural killer cells and cytotoxic T lymphocytes in vivo.

We have previously detected perforin expression in a subpopulation of asialo GM1+ natural killer (NK) cells and CD8+ T lymphocytes in murine spleen cells by immunocytochemical staining with an anti-perforin monoclonal antibody. In the present study, more detailed analyses of perforin expression in murine cytotoxic lymphocyte subpopulations were performed. The expression of perforin in asialo GM1+ spleen cells was predominantly confined to the NK1.1+ subset, where all NK activity also resided. Perforin expression was also studied on alloreactive cytotoxic T lymphocyte (CTL) induced in vivo. The cells expressing perforin in peritoneal exudate lymphocytes predominantly resided in the CD8+ T cell subpopulation co-expressing asialo GM1 where an allospecific CTL activity also resided. Furthermore, the percentage of perforin-positive cells in this population was greatly reduced after stimulation with anti-CD3 or anti-T cell receptors antibodies, which induce serine esterase release from the cytoplasmic granules. These findings highly suggest that perforin is involved in in vivo NK cell- and CTL-mediated cytolysis.

Animals↗
PubMed1992

Perforin is expressed in CTL populations generated in vivo.

Immunization of C57BL/6 mice with syngeneic tumor cells, MBL-2, resulted in the generation of antitumor effector cells in vivo. The immunized C57BL/6 mice permanently rejected viable MBL-2 lymphoma cells, but not B16 melanoma cells. Cytotoxic T cells obtained from MBL-2-immunized mouse peritoneal cells (PEC) showed specific cytotoxicity against MBL-2, but not to YAC-1, RDM-4 and Meth A cells. By sorting with FACStar, the specific CTL were characterized as TCR alpha beta+ CD8+ T cells. Moreover, the cytoplasm of in vivo-induced CTL was stained with a monoclonal antibody against perforin. The localization of perforin in cytoplasmic granules of CTL was demonstrated by electron microscope analysis. This experiment presented the first evidence that in vivo-induced CD8+ CTL against syngeneic tumor cells expressed significant amounts of perforin.

Animals↗
PubMed1992

Expression of perforin and cytolytic potential of human peripheral blood lymphocyte subpopulations.

To verify the physiological role of the pore-forming protein perforin in vivo, its expression in subpopulations of human peripheral blood lymphocytes was examined by immunocytochemical staining and their cytolytic potentials compared. In addition to NK cells and gamma delta T cells, which uniformly expressed abundant perforin in their cytoplasmic granules, only a small subpopulation of CD8+ alpha beta T cells contained perforin, namely the CD11b+ subset. However, in vitro activation with an anti-CD3 antibody and IL-2 induced perforin expression in approximately 50% of the CD8+CD11b- T cells and also in a small subset of CD4+ T cells. A distribution of perforin in CD8+ and CD4+ T cells, similar to in vitro activated T cells, was observed in fresh peripheral blood lymphocytes from infectious mononucleosis patients. In all instances, the expression of perforin correlated with the cytolytic potential of these subpopulations. The results strongly suggest that perforin plays a role in the manifestation of cytotoxic activity in vivo.

Antigens, CD↗
PubMed1992

Cellular and molecular bases for the immunopathology of the myocardial cell damage involved in acute viral myocarditis with special reference to dilated cardiomyopathy.

Cell-mediated autoimmunity has been strongly implicated in the pathogenesis of the myocardial cell damage involved in viral myocarditis. To investigate the cellular and molecular bases of both target cells and effector cells for cell-mediated cytotoxicity involved in viral myocarditis and dilated cardiomyopathy, we first examined the expression of major histocompatibility complex (MHC) antigens and a cell adhesion molecule, intercellular adhesion molecule-1 (ICAM-1) in myocardial cells of a murine model of viral myocarditis and in patients with acute myocarditis and dilated cardiomyopathy. Secondly, we analyzed the characteristics of the infiltrating cells in the heart, especially the expression of a cytolytic factor, perforin. We found that Coxsackievirus B3 (CVB3)-induced murine acute myocarditis resulted in enhanced expression of MHC (class I) antigens and ICAM-1 on myocardial cells, and that perforin was abundantly expressed in NK (natural killer)-like large granular lymphocytes (LGL), which represent the main infiltrating cell type in the early stage. Immunoelectron microscopic study showed killer cells directly damaging cardiac myocytes by the release of perforin. Perforin was also expressed in the infiltrating cells in the heart of a patient with acute myocarditis. Both MHC antigens and ICAM-1 were clearly expressed in the hearts of patients with acute myocarditis and dilated cardiomyopathy. These data provided direct evidence that cell-mediated cytotoxicity plays a critical role in the myocardial cell damage involved in viral myocarditis.

Acute Disease↗
PubMed1991

Antigen-specific directional target cell lysis by perforin-negative T lymphocyte clones.

Despite a number of reports indicating that perforin, a pore-forming protein, is the primary effector molecule mediating specific target cell lysis by cytotoxic T lymphocytes (CTL), several lines of evidence suggest the existence of perforin-independent mechanisms. We established class II-restricted, soluble protein-specific CD4+ T cell clones with killing function which do not express a detectable amount of perforin and perforin mRNA. Nevertheless, these clones induced cytolysis and DNA fragmentation of target cells in a specific and highly directional manner which was not inhibitable by antibody against TNF/lymphotoxin. These data not only indicate the existence of cytotoxic T cell subsets which do not utilize perforin, but also suggest that perforin is not mandatory for specific target lysis by T cells.

Animals↗
PubMed1991

Expression of perforin in infiltrating cells in murine hearts with acute myocarditis caused by coxsackievirus B3.

BACKGROUND: Cell-mediated autoimmunity has been strongly implicated in the pathogenesis of viral myocarditis. METHODS AND RESULTS: Using a murine model of acute myocarditis caused by coxsackievirus B3, we analyzed the phenotypes and morphology of the infiltrating cells in the hearts by immunofluorescence and electron microscopy. We also examined the expression of a cytolytic factor, perforin, in the infiltrating cells by immunoperoxidase and in situ hybridization. We found that the dominant population of the infiltrating cells were asialo GM1 positive, were negative for T-cell markers, and had electron-dense cytoplasmic granules, which is consistent with a morphology of large granular lymphocytes. Perforin was found in the cytoplasmic granules of the infiltrating cells expressing perforin messenger RNA. These findings provide for the first time the direct evidence that the first wave of cell infiltration in hearts mainly consists of killer cells and strongly suggests that perforin plays, in part, an important role in myocardial cell damage involved in acute viral myocarditis. T-helper cells and cytotoxic T lymphocytes made up the second wave of cell infiltration. CONCLUSIONS: As we previously reported, the expression of major histocompatibility complex class I antigen on cardiac myocytes induced by the infiltrating cells, such as killer cells, may facilitate the interaction between cardiac myocytes and cytotoxic T lymphocytes, and may lead to further myocardial cell damage in a later phase.

Acute Disease↗
PubMed1990

Constitutive expression of pore-forming protein in peripheral blood gamma/delta T cells: implication for their cytotoxic role in vivo.

The cytotoxic activity and pore-forming protein (PFP) expression of human peripheral blood (PB) gamma/delta T cells were examined. Fresh gamma/delta T cells isolated from PB lymphocytes by fluorescence-activated cell sorting exhibited a substantial natural killer-like cytotoxic activity against K562 target cells and had a high cytotoxic potential triggered by anti-CD3 monoclonal antibody (mAb) against P815 target cells bearing Fc gamma R. Immunocytochemical staining with an anti-PFP mAb revealed that virtually all PB gamma/delta T cells are granular lymphocytes with abundant PFP in their cytoplasmic granules. Constitutive expression of PFP in PB gamma/delta T cells was also demonstrated by Northern blot analysis. These observations support the proposed role of gamma/delta T cells in cytolytic immune surveillance in vivo.

Antibodies, Monoclonal↗
PubMed1990

Interleukin 2 induction of pore-forming protein gene expression in human peripheral blood CD8+ T cells.

Our studies have analyzed pore-forming protein (PFP) mRNA expression in resting and stimulated human peripheral blood CD3- large granular lymphocytes (LGL), CD3+ T cells, and their CD4+ or CD8+ subsets. Signals that stimulate T cells to develop cytotoxic activity (i.e., IL-2 or OKT-3 mAb) led to the induction of PFP mRNA in T cells. The data indicated that IL-2 directly increased PFP mRNA in the CD8+ subset of T cells, in the absence of new DNA or protein synthesis. Abrogation of IL-2-induced PFP mRNA expression and cytotoxic potential of T cells by the anti-p75 IL-2 receptor mAb suggested that low numbers of p75 IL-2 receptors on CD8+ T cells were capable of transducing signals responsible for these IL-2-induced effects. The induction of T cell PFP mRNA via CD3, using OKT-3 mAb, was less rapid but greater than that caused by IL-2; however, a combination of PMA and ionomycin, which bypasses crosslinking of the TCR/CD3 complex, could not mimic this increase in PFP mRNA levels in T cells. The role of second messenger systems in regulating PFP mRNA expression remains to be determined. In contrast, high constitutive PFP mRNA expression was observed in CD3- LGL and these mRNA levels could not be enhanced by stimulation with IL-2. The cytotoxic potential of peripheral blood T cells and LGL induced in response to IL-2 correlated with IL-2-induced PFP mRNA levels in these cells and was consistent with PFP being one of several important molecules involved in the effector function of cytotoxic lymphocytes.

Antibodies, Monoclonal↗
PubMed1990

Perforin gene expression in granular lymphocyte proliferative disorders.

By Northern blot analysis using a cDNA clone of the perforin gene, we studied the levels of perforin mRNA in peripheral blood mononuclear cells from 11 cases of granular lymphocyte-proliferative disorders (GLPDs). The granular lymphocytes studied were characterized by morphologic, immunophenotypic, and immunogenotypic analyses. Cytolytic functions of the lymphocytes assayed included nonmajor histocompatibility complex-requiring cytotoxicity, anti-CD3-redirected cytotoxicity, antibody-dependent cellular cytotoxicity, and lectin-dependent cellular cytotoxicity. The results showed that in lymphocytes with strong cytolytic functions high levels of perforin mRNA existed, whereas in lymphocytes with weak or undetectable levels of cytolytic functions, low levels of perforin mRNA existed. Because the levels of perforin mRNA correlated with those of cytolytic functions, perforin is probably a mediator in cytolytic functions of granular lymphocytes in patients with GLPDs. When the lymphocytes were cultured for 1 day, however, the levels of cytolytic activity were increased, and those of perforin mRNA were decreased. Therefore, we cannot rule out the possibility that factors other than perforin protein are involved in the cytolytic functions of granular lymphocytes.

Antigens, CD↗
PubMed1990

Thy-1-positive dendritic epidermal cells contain a killer protein perforin.

The killer cell characteristics of Thy-1-positive dendritic epidermal cells (Thy-1+ DEC) were examined. Four Thy-1+ DEC clones which were established from athymic nude mice exhibited spontaneous or lectin-redirectable cytotoxic activity against some murine tumor cell lines in a 4 h 51Cr-release assay. A colorimetric assay for benzyloxycarbonyl-L-lysine-thiobenzyl ester esterase revealed a strong serine esterase activity expressed in all cell clones. In addition, Northern blot analysis using a murine perforin cDNA probe revealed that all four Thy-1+ DEC clones expressed abundant mRNA for perforin, as do most killer T cells. More importantly, immunocytochemical staining with an anti-perforin monoclonal antibody revealed that not only all four Thy-1+ DEC clones but also a part of freshly isolated Thy-1+ DEC from normal and nude mice contained perforin. These results demonstrate that Thy-1+ DEC exhibit typical killer cell characteristics in vitro and in vivo. These data also suggest that Thy-1+ DEC may play a cytotoxic role in protecting the integrity of skin from infection or neoplastic transformation.

Animals↗
PubMed1990

Perforin, a pore-forming protein detectable by monoclonal antibodies, is a functional marker for killer cells.

Perforin is one of the important cytolytic factors in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In this paper, we report rat mAbs against mouse perforin established by immunization with a recombinant mouse perforin fragment. These mAbs reacted with purified mouse perforin prepared from cytoplasmic granules of an NK-like cell line in ELISA and Western blot analysis. However, none of these mAbs blocked the hemolytic activity of mouse perforin or absorbed it when fixed in the solid phase. These results indicate that all of these mAbs react with denatured but not with native mouse perforin. By using a combination of the mAbs, we established a sandwich ELISA, for quantitating the cellular contents of perforin. These mAbs were also useful for immunohistochemical staining analysis, and perforin was detected in the cytoplasmic granules of CTL and NK cell lines. Perforin was also detected in a minor population of lymphocytes of the spleen, liver, and lymph node. In normal spleen cells of 5- to 8-week-old mice, 12-15% of asialo GM1+ cells and 7-21% of CD8+ T cells were perforin-positive, but CD4+ T cells, B cells, and macrophages were totally negative. These data clearly show that perforin is expressed in cells of a cytotoxic character in normal mice, in the same way as in primed mice.

Animals↗
PubMed1990

Antimouse laminin antibodies in IgA nephropathy and various glomerular diseases.

IgG, IgA and IgM class antibodies to mouse laminin and human fibronectin in sera from patients with various glomerular diseases (50 cases of IgA nephropathy, 5 cases of minimal-change nephrotic syndrome; 6 cases of membranous nephropathy, 5 cases of systemic lupus erythematosus, 2 cases of Henoch-Schönlein purpura, 3 cases of poststreptococcal nephritis and 4 cases of preeclampsia) and from 30 normal controls were tested using a solid-phase enzyme-linked immunosorbent assay method. IgA antimouse laminin antibody titers in sera from IgA nephropathy patients were significantly higher (p less than 0.05) than in controls. There were no statistical differences in IgA antimouse laminin antibody titers between patients with other glomerular diseases and normal controls. IgM antimouse laminin antibody was significantly raised (p less than 0.01) in sera from patients with preeclampsia. The reaction of mouse laminin with the IgA nephropathy and preeclampsia sera on each of the IgA and IgM assay systems was inhibited by the antigen at up to 5 micrograms/ml. However, it was not inhibited by anti-C3d, anti-C1q, anti-J chain and antisecretory component sera or saccharides. The reaction of mouse laminin with an exceptionally high-titer IgA antimouse laminin antibody serum from a normal control on the IgA assay system was clearly inhibited by 1 mM of melibiose, which contains alpha-galactosyl residues. The same concentration of melibiose, however, did not inhibit the reaction of mouse laminin with IgA nephropathy sera on the same assay system. Treatment of mouse laminin with alpha-galactosidase did not alter any binding from IgA nephropathy sera but binding was lost from an exceptionally high-titer normal control serum. There were no correlations between serum IgA level and IgA antimouse laminin antibody titer in sera from IgA nephropathy patients. Immunoblot techniques revealed the presence of antibody in sera from IgA nephropathy patients reacting with both subunits A and B of laminin, somewhat stronger with laminin A. None of the sera tested contained antifibronectin antibodies. These results indicate that the IgA antimouse laminin antibody is a specific antibody in IgA nephropathy and might play a role in the pathogenesis of the nephritis since mouse laminin and human mesangial laminin present a common epitope.

Adolescent↗
PubMed1989

Molecular cloning of rat cytolysin.

Rat cytolysin is one of the cytolytic factors present in the cytoplasmic granules of rat NK-like cytolytic cells and purified cytolysin exhibits an apparent Mr or 70 kDa. Cytolysis produced by cytolysin occurs in the presence of Ca2+ and is accompanied by the formation of membrane lesions of 160 A diameter. We have isolated a cDNA encoding rat cytolysin from the cDNA library of a rat large granular lymphocyte (LGL) cell line, by hybridization of the rat library with a cDNA probe for mouse perforin. The amino acid sequence deduced from the nucleotide sequence of the isolated cDNA insert indicates that the mature cytolysin protein consist of 534 amino acids with a leader peptide of 20 amino acids. The protein contains two functionally important domains: the first domain is believed to contain the transmembrane channel and the second domain consists of an epidermal growth factor-type "class B" cysteine-rich region. A comparison with mouse perforin indicates that the two genes are very similar (89.9% nucleotide and 84.9% amino acid identity). Northern blot hybridization analysis indicates that cytolysin mRNA is expressed in rat lymphocytes (lymphokine-activated killer cells and LGL cells) and LGL cell lines.

Amino Acid Sequence↗
PubMed1989

Molecular cloning and chromosomal assignment of a human perforin (PFP) gene.

Human perforin cDNA was isolated and the complete nucleotide sequence of the gene determined. The deduced amino acid sequence of human perforin showed 68.4% similarity to that of mouse perforin. RNA blot analysis of the human perforin gene revealed that the gene product is expressed preferentially in killer-type cells among cell lines tested, and in large granular lymphocytes among the peripheral blood mononuclear cells. In situ hybridization analysis with a human perforin cDNA probe revealed that the human perforin (PFP) gene is located on chromosome 17q11-21.

Base Sequence↗
PubMed1988

Homology of perforin to the ninth component of complement (C9).

Perforin is one of the cytolytic factors present in the cytoplasmic granules of mouse cytotoxic T lymphocytes and natural killer cells. We have determined the sequence of the N-terminal amino acids of perforin purified from a mouse natural killer cell line, and, by using oligonucleotide probes corresponding to the amino acid residues, we have identified a complementary DNA encoding perforin from the cDNA library of a mouse cytotoxic T lymphocyte clone. As predicted from the functional similarities between perforin and the ninth component of the serum cytolytic system, complement (C9) (refs 4-8), the deduced primary structure of perforin has homology with C9 at their respective functionally conserved regions. We find that perforin is only expressed in killer cell lines, and not in helper T lymphocytes or other tumour cells tested. Thus we have provided direct molecular evidence that a killer-cell-specific protein evolutionally linked to C9 is involved in cell-mediated cytolysis.

Amino Acid Sequence↗