SEARCH · Search PubMed

Results for “Perforin”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 343 records · Page 19Linked to original sources

PubMed2006

NK cells rapidly remove B16F10 tumor cells in a perforin and interferon-gamma independent manner in vivo.

Natural killer (NK) cells have been shown critical in reducing tumor lung metastasis in various murine cancer models. Effector molecules such as perforin and IFN-gamma may play important roles in inhibition of metastasis. However, most of these conclusions were based on experiments that involved quantitation of metastatic colonies several weeks after tumor challenge. The roles of NK cells and their effector molecules (perforin and IFN-gamma) in the initial immune responses against tumor metastasis in lungs are still unknown. By using the B16F10 melanoma tumor model combined with confocal microscopy, we observed an increase in numbers of B16F10 cells in NK-depleted mice at 60 min post tumor inoculation, but this effect was independent of perforin or IFN-gamma. In addition, NK cell numbers in lungs after tumor injection rapidly increased suggesting a redistribution of NK cells in the lungs. However, NK cells were not found in contact with tumor cells until day 6 or later. Our data indicate that during early responses against B16F10 cells, NK cells use another mechanism(s) besides perforin and IFN-gamma to prevent tumor metastasis.

Animals↗

PubMed1997

Role of perforin, granzymes and the proliferative state of the target cells in apoptosis and necrosis mediated by bispecific-antibody-activated cytotoxic T cells.

Bispecific monoclonal antibodies (bi-mAb), directed against a tumor-associated antigen and the CD3 or CD28 antigen on T lymphocytes, induce activation of resting T lymphocytes and target-specific tumor cell lysis. We now show that both necrosis and apoptosis contribute to T-cell-mediated tumor cell destruction. Even though T cells up-regulate FAS/APO-1 expression upon bi-mAb stimulation, FAS/APO-1-mediated apoptosis does not contribute to bi-mAb-mediated destruction of Hodgkin's cells. CD8+ lymphocytes were the most potent effectors of bi-mAb-mediated cytotoxicity and had the highest levels of mRNA coding for perforin and granzyme A and B. Ca2+-complexing agents, which abrogate perforin activity, led to decreased levels of necrosis, while inhibition of granzyme activity in effector or target cells had a similar effect on apoptosis. Granzyme-mediated apoptosis critically dependent on the proliferative state of the target cells, while perforin-induced necrosis was not cell-cycle-dependent. Our results underline the importance of the expression levels of perforin and granzymes in the effector T cells and of the proliferative state of the target cells in bi-mAb-mediated apoptosis and necrosis of tumor cells.

Antibodies, Bispecific↗

PubMed1991

A microassay for the pore-forming activity of complement, perforin, and other cytolytic proteins based on confocal laser scanning microscopy.

A fluorescence microscopic assay for the activity of complement, perforin, and other cytolytic proteins which form transmembrane pores in cellular membranes is described. The assay was worked out and tested with red blood cell membranes (ghosts) and was then applied to intact hemoglobin-free cells. Resealed human erythrocyte ghosts were incubated with complement or perforin. A small polar fluorescent probe (fluorescein-labeled 1-kDa dextran, FD1) which permeates through complement and perforin pores but not through normal cell membranes was added to the samples. The capability of the confocal laser scanning microscope (CLSM) to generate thin optical sections was exploited to visualize and quantitate fluorescence inside single ghosts and thus determine the fraction of ghosts which had become permeable for FD1. The activity of complement or perforin was quantitated by plotting the fraction of permeable cells versus the concentration of the pore-forming protein. The results were in good agreement with those of a conventional hemolytic assay. The CLSM-based assay was then applied to intact hemoglobin-free cells for which only few alternative assays are available. Compared to conventional hemolytic assays for the activity of pore-forming proteins the assay described here can be applied to a large variety of natural and artificial membrane systems. The assay can be performed under nonlysing conditions. Furthermore, the assay is simple, relatively fast, and requires only extremely small amounts of cells and pore-forming proteins.

Animals↗

PubMed1987

Inhibition of the lytic activity of perforin (cytolysin) and of late complement components by proteoglycans.

The complement components (C6, C7, C8 and C9) implicated in the lysis of target cells and the pore-forming, lytic protein from cytotoxic T-lymphocytes and NK-cells, perforin, contain an amino acid sequence which is highly homologous to a repeat unit identified in the LDL-receptor (Tschopp et al., 1986, Nature, 322, 831-834). The domain of the LDL-receptor, which is thought to interact with a positively charged segment of its ligands apoprotein B and E, is rich in cysteine residues and contains a cluster of negative charges. We show that the negatively charged molecules suramin and glycosaminoglycans, the positively charged peptides protamine and polylysine, all of which are known to abolish binding of LDL to its receptor (Goldstein et al., 1985, A. Rev. cell. Biol., 1, 1-39) inhibit the lytic activities of C6, C7, C8, C9 and perforin. Moreover, these negatively charged molecules are potent inhibitors of cytolytic T-lymphocyte-mediated lysis of target cells, suggesting a functionally crucial role for perforin in cell-mediated cytolysis. We propose that the negatively charged, cysteine-rich domain of these complement proteins and perforin interacts with an as yet unidentified positively charged segment of its ligand in a manner analogous to the LDL-LDL receptor interaction. Homologous cysteine-rich domains in functionally unrelated proteins may therefore be functionally conserved as ideal rigid interaction domains with the conserved cysteine residues as framework. Specificity of the domain for its ligand would be conferred by the non-conserved amino acid residues.

Cell Line↗

PubMed1995

Perforin: structure and function.

Perforin is a cytolytic mediator produced by killer lymphocytes, and is stored in and released by cytoplasmic granules. The protein is partially homologous to the terminal components of the membrane attack complex of complement and produces pores of up to 20 nm in diameter on target membranes. Its genomic and protein structures have recently been unraveled, and its function elucidated through the availability of genetically engineered, perforin-deficient mice. Here Chau-Ching Liu, Craig M. Walsh and John Ding-E Young briefly outline certain biochemical and molecular features of perforin, and discuss the still-evolving issues concerning the relevance of perforin and Fas in cell killing.

Animals↗

PubMed1992

Human perforin (PRF1) maps to 10q22, a region that is syntenic with mouse chromosome 10.

Perforin (PRF1) is a cytolytic, channel-forming protein of cytolytic T cells, natural killer cells, and granulated metrial gland cells and plays a crucial role in the killer cell-mediated elimination of virally infected host cells, tumor cells, and allotransplants. Two-thirds of the perforin sequence is homologous to the lytic, channel-forming complement proteins C6, C7, C8 alpha, C8 beta, and C9. Using cosmid DNA containing the PRF1 gene as a probe for fluorescence in situ hybridization, we have reevaluated its chromosomal location. Previously assigned to chromosome 17q11-q21, it has now been mapped to 10q22. The human PRF1 locus lies within a conserved synteny segment present on mouse chromosome 10, consistent with the previous chromosomal assignment of mouse perforin. The perforin locus is not linked to any of the genes of the terminal complement system.

Animals↗

PubMed2005

Perforin triggers a plasma membrane-repair response that facilitates CTL induction of apoptosis.

Perforin delivers granzymes to induce target-cell apoptosis. At high concentrations, perforin multimerizes in the plasma membrane to form pores. However, whether granzymes enter target cells via membrane pores is uncertain. Here we find that perforin at physiologically relevant concentrations and during cell-mediated lysis creates pores in the target-cell membrane, transiently allowing Ca(2+) and small dyes into the cell. The Ca(2+) flux triggers a wounded membrane-repair response in which internal vesicles, including lysosomes and endosomes, donate their membranes to reseal the damaged membrane. Perforin also triggers the rapid endocytosis of granzymes into large EEA-1-staining vesicles. The restoration of target-cell membrane integrity by triggering the repair response is necessary for target cells subjected to cytotoxic T lymphocyte attack to avoid necrosis and undergo the slower process of programmed cell death. Thus, the target cell actively participates in determining its own fate during cell-mediated death.

Apoptosis↗

PubMed2007

Chromosome transfer activates and delineates a locus control region for perforin.

Perforin gene (PRF1) transcription regulates perforin expression in NK cells and CTL. Here we identified the locus-wide ensemble of cis-acting sequences that drives PRF1 transcription physiologically. By using chromosome transfer, we revealed that de novo activation of a silent PRF1 locus was controlled by a 150 kb domain comprised of 16 DNase I hypersensitive sites (DHSs). These cis-acting sequences included a locus control region (LCR) and conferred developmentally appropriate and lineage-specific expression of human perforin from BAC transgenes. The LCR included four distal DHSs that were required for perforin expression from its natural locus, and their engineered deletion from the PRF1 BAC transgene abolished LCR function and led to rapid gene silencing. Thus, LCR function is central for regulating the developmental and activation-specific PRF1 promoter activity characteristic of NK cells and CTL.

Animals↗

PubMed2006

Protection against polyoma virus-induced tumors is perforin-independent.

CD8 T cells are necessary for controlling tumors induced by mouse polyoma virus (PyV), but the effector mechanism(s) responsible have not been determined. We examined the PyV tumorigenicity in C57BL/6 mice mutated in Fas or carrying targeted disruptions in the perforin gene or in both TNF receptor type I and type II genes. Surprisingly, none of these mice developed tumors. Perforin/Fas double-deficient radiation bone marrow chimeric mice were also resistant to PyV-induced tumors. Anti-PyV CD8 T cells in perforin-deficient mice were found not to differ from wild type mice with respect to phenotype, capacity to produce cytokines or maintenance of memory T cells, indicating that perforin does not modulate the PyV-specific CD8 T cell response. In addition, virus was cleared and persisted to similar extents in wild type and perforin-deficient mice. In summary, perforin/granzyme exocytosis is not an essential effector pathway for protection against PyV infection or tumorigenesis.

Animals↗

PubMed2004

Perforin mediates endothelial cell death and resultant transplant vascular disease in cardiac allografts.

T cell-induced endothelial injury is an important event in the development of transplant vascular disease (TVD), the leading expression of chronic rejection of vascularized organ transplants. However, the precise contribution of perforin to vascular damage in allografts and resultant TVD has not been addressed in vivo. Minor histocompatability antigen mismatched mouse heterotopic cardiac transplants were performed from 129J donors into C57Bl/6 (wild-type (WT)) or perforin knockout (PKO) recipients. Perforin was abundant in immune infiltrates in the myocardium and vasculature of transplanted hearts in WT mice. Allograft coronary arteries in both WT and PKO mice had considerable vasculitis. There was also marked endothelial disruption, as well as TUNEL-positivity in the endothelial region, in coronary arteries of hearts transplanted into WT mice that was not evident in PKO recipients (P = 0.05). At 30 days post-transplantation, intimal thickening was assessed on elastic Van Gieson-stained ventricular sections. There was an average of 54.2 +/- 6.7% luminal narrowing of coronary arteries in allografts from WT mice as compared to 13.4 +/- 5.1% luminal narrowing in PKO counterparts (P < 0.00002). In summary, perforin plays a primary role in endothelial damage and the resultant onset and progression of TVD.

Animals↗

PubMed2001

A comparative study of the expression of cytotoxic proteins in allergic contact dermatitis and psoriasis: spongiotic skin lesions in allergic contact dermatitis are highly infiltrated by T cells expressing perforin and granzyme B.

Recent reports indicate that cytotoxic T cells are critically involved in contact hypersensitivity reactions in animals. In this study we sought to investigate the in vivo expression of cytotoxic granule proteins in the elicitation phase of allergic contact dermatitis in humans. Skin biopsy specimens were obtained from patients with allergic contact dermatitis (n = 8) and psoriasis (n = 6) and from controls with normal skin (n = 6). Expression of perforin and granzyme B was investigated by in situ hybridization and immunohistochemistry. In contrast to normal skin and psoriasis, a significant enhancement of perforin and granzyme B gene expression and immunoreactivity was observed in the mononuclear cell infiltrate of allergic contact dermatitis. Immunoreactivity for perforin and granzyme B was mainly found in the cytoplasm of lymphocytic cells, which were located in the dense perivascular infiltrate as well as at sites of marked spongiosis in the epidermis. Double immunostaining revealed that both CD4+ and CD8+ T cells are capable of expressing perforin and granzyme B. In conclusion, our data suggest that T-cell-mediated mechanisms involving cytotoxic granule proteins may elicit epidermal cell injury in vivo and thereby strongly contribute to the development of allergic contact dermatitis in humans.

Adult↗

PubMed2001

Full-length cloning and 3'-terminal portion expression of human perforin cDNA.

BACKGROUND: Perforin (also known as pore-forming protein, PFP) is one of the main effector molecules which natural killer cells (NK) and cytotoxic T lymphocytes (CTL) utilize to kill their targets both in vivo and in vitro. We report the full length of human perforin cDNA, which was cloned from liver tissue. RESULTS: Sequencing analysis showed that there were discrepancies of four nucleotides and three amino acids compared with previously published sequence of human PFP. The cDNA fragment was then inserted into fusion protein expressive vector pGEX-2T to construct a recombinant expressive plasmid. The C-terminal truncated 125 amino acids polypeptide (410-534aa) of human perforin (hPFP-C) was selectively expressed in a form of fusion protein. Under the induction of IPTG, GST/hPFP-C fusion protein was expressed in E. coli BL21 (DE3). The fusion protein GST/hPFP-C was purified by affinity chromatography with glutathione agarose. The recombinant hPFP-C obtained by thrombin cleavage showed a significant hemolytic activity when tested with rabbit erythrocytes. CONCLUSION: These results suggest that the domain responsible for lytic function lies not only in the N-terminal portion but also in the C-terminal portion of perforin molecule. The recombinant hPFP-C protein will be useful as a highly purified biological factor for immunological, pathological and structural studies.

Amino Acid Sequence↗

PubMed1998

Lymphokine-activated killer cells induced in vivo in mice showing IL-2 toxicity have cytoplasmic granules containing perforin and its hemolytic activity.

Cytoplasmic granules of LAK cells isolated from mice showing side effects of recombinant human IL-2 (rhIL-2) display BLT-serine esterase activity and calcium-dependent hemolytic activity and seem to be involved in the mechanisms of LAK cell-mediated rhIL-2 toxicity. In this report, the hemolytically active substance was partially purified from the LAK granules and shown to be mouse perforin. Mice were implanted with miniosmotic pumps filled with a dose of rhIL-2 that induced known toxicity. The LAK granules were isolated from LAK cell-rich subcutaneous tissue around the infusion sites. The hemolytically active substance in the granule preparation was solubilized by 2 M NaCl and passed through a butyl Sepharose column and then a DEAE Toyopearl column. In immunoblotting, the hemolytically active fractions reacted with an anti-mouse perforin mAb and the reaction depended on the activity. The molecular sizes of the perforin-positive bands were 66 kDa and 51 kDa under reducing and nonreducing conditions, respectively. These results confirmed the existence of mouse perforin and its hemolytic activity in the LAK granules of rhIL-2-treated mice, as has been shown for granules of cytotoxic T or NK cell clones in vitro.

Animals↗

PubMed2003

Perforin and the granule exocytosis cytotoxicity pathway.

Perforin defects have been identified in humans with familial hematophagocytic lymphohistiocytosis. The pathology of these patients has dramatically illustrated an under-appreciated role for perforin in the regulation of T-cell responses in vivo, and experimental studies are shedding light on the mechanisms involved. The detailed molecular mechanisms of perforin's mandatory role in the cytotoxic T lymphocyte (CTL)-mediated granule exocytosis death pathway and of granzyme entry into target cells remain unclear. In model systems measuring apoptosis by granzyme B and sublytic perforin, pore formation is undetectable during granzyme entry. Selfprotection of cytotoxic lymphocytes after degranulation can be explained by surface expression of the granule protease cathepsin B, as shown by suicidal degranulation in the presence of specific inhibitors.

Animals↗

PubMed2003

Perforin is required for innate and adaptive immunity induced by heat shock protein gp96.

Tumor-secreted gp96-Ig is highly immunogenic and triggers CD8 T cell-mediated tumor rejection. In vivo secreted gp96-Ig and gp96-myc cause NK activation and clonal expansion of specific CD8(+) CTL in wild-type and in Fas-ligand-deficient (gld) mice but not in perforin- (PKO) or IFN-gamma-deficient (GKO) mice. Transfer of perforin-competent NK cells restores the ability of PKO mice to clonally expand CD8 CTL in response to gp96-Ig. The data demonstrate an essential role for perforin-mediated functions in the activation of innate and adaptive immunity by heat shock protein gp96-peptide complexes. Crosspresentation of antigens by heat shock proteins seems to require a perforin-dependent positive feedback loop between NK and DC for both sustained NK activation and clonal CTL expansion. The studies also explain how depressed NK activity in patients with tumors or after viral infections could diminish CTL responses.

Adoptive Transfer↗

PubMed2005

NK cells promote islet allograft tolerance via a perforin-dependent mechanism.

Although major histocompatibility complex (MHC) class II-restricted CD4 T cells are well appreciated for their contribution to peripheral tolerance to tissue allografts, little is known regarding MHC class I-dependent reactivity in this process. Here we show a crucial role for host MHC class I-dependent NK cell reactivity for allograft tolerance in mice induced through either costimulation blockade using CD154-specific antibody therapy or by targeting LFA-1 (also known as CD11a). Tolerance induction absolutely required host expression of MHC class I, but was independent of CD8 T cell-dependent immunity. Rather, tolerance required innate immunity involving NK1.1(+) cells, but was independent of CD1d-restricted NKT cells. Therefore, NK cells seem to be generally required for induction of tolerance to islet allografts. Additional studies indicate that CD154-specific antibody-induced allograft tolerance is perforin dependent. Notably, NK cells that are perforin competent are sufficient to restore allograft tolerance in perforin-deficient recipients. Together, these results show an obligatory role for NK cells, through perforin, for induction of tolerance to islet allografts.

Animals↗

PubMed2006

Perforin-mediated target-cell death and immune homeostasis.

The granule exocytosis pathway of cytotoxic lymphocytes is crucial for immune surveillance and homeostasis. The trafficking of granule components, including the membrane-disruptive protein perforin, to the immunological synapse leads to the delivery of granule proteases (granzymes) into the target cell and its destruction through apoptosis. Several independent molecular abnormalities associated with defects of either granule trafficking or perforin function can cause cytotoxic lymphocyte dysfunction. In humans, inherited perforin mutations result in severe immune dysregulation that manifests as familial haemophagocytic lymphohistiocytosis. This Review describes recent progress in defining the structure, function, biochemistry and cell biology of perforin.

Animals↗

PubMed1990

Reversible cell damage by T-cell perforins. Calcium influx and propidium iodide uptake into K562 cells in the absence of lysis.

The non-lethal effects of the lymphocyte-derived pore-forming toxin perforin on the human erythroleukaemia cell line K562 were investigated. By using the fluorescent Ca2+ indicator fura-2, perforin was shown to cause intracellular Ca2+ concentration to rise transiently into the micromolar range in the absence of cell death. By fluorescence-activated cell sorting it was demonstrated that K562 cells took up the membrane-impermeant nuclear stain propidium iodide (PI) when exposed to non-lethal doses of perforin. The permeability to PI was short-lived, confirming the transience of the perforin pore. Analogies with non-lethal effects and recovery processes occurring in nucleated cells exposed to the membrane-attack complex of complement are drawn.

Animals↗