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PubMed1997

Epidermal apoptotic cell death in erythema multiforme and Stevens-Johnson syndrome. Contribution of perforin-positive cell infiltration.

OBJECTIVE: To clarify the mechanism of epidermal cell death in erythema multiforme. DESIGN: Retrospective study. SETTING: Academic referral center. PARTICIPANTS: Nine patients with Stevens-Johnson syndrome (SJS), 9 patients with Hebra disease (EMH), and 5 healthy volunteers. INTERVENTIONS: Biopsy specimens were obtained from the border of the fresh lesions before treatment. Control specimens were obtained from normal skin. MAIN OUTCOME MEASURES: Histopathological epidermal apoptosis detected with nuclear DNA fragmentation and counts of dermal immunoreactive perforin-positive infiltrates were compared between SJS and EMH. RESULTS: Eight patients (89%) with SJS showed clear apoptosis with keratinocyte DNA fragmentation. All SJS samples had intensive perforin-positive dermal infiltrates. Only 3 patients (33%) with EMH showed apoptotic change, and it was to a far less extent with far less dermal perforin-positive infiltrates. Control specimens showed no apoptotic cells in the epidermis or expression of perforin in the dermis. CONCLUSIONS: Perforin mediates apoptosis in the pathogenesis of the epidermal cell changes in SJS but not in EMH. In addition to the differences in clinical severity and histopathological conditions, our findings indicate a pathogenic difference between SJS and EMH.

Antibodies, Monoclonal↗
PubMed1998

Arrest of the cell cycle reduces susceptibility of target cells to perforin-mediated lysis.

Cytotoxic T lymphocytes secrete a pore-forming cytolysin, perforin, that damages membranes of target cells. They also ligate Fas receptors on target cells and provoke apoptotic death. A20 (B lymphoma) and P815 (mastocytoma) cell lines were examined for their susceptibility to perforin-mediated lysis and to Fas-induced apoptosis after blockade of the cell cycle at the G1/S interface. Cells were arrested at the G1/S interface by inhibition of DNA synthesis with thymidine or aphidicolin. Subsequently, the treated cells were incubated either with CTL cytotoxic granules or the Fas-specific monoclonal antibody Jo-2. We show that arrest of the cell cycle at the G1/S interface markedly reduced the susceptibility of target cells to perforin-mediated lysis. In contrast, growth arrest with thymidine or aphidicolin increased susceptibility of A20 and P815 cells to Fas-mediated apoptosis. Susceptibility to lysis by intact CTLs was not affected significantly by blockade of target cells with aphidicolin or thymidine. When cells surviving exposure to perforin-containing granules were isolated on Ficoll density gradients and cell-cycle profiles were examined by flow cytometry, the ratio of G1 to G2 cells increased among the survivors exposed to granules in contrast to controls incubated with buffer alone. The data suggest that cells in G1 phase of the cell cycle are less susceptible to the perforin pathway than cells in G2 and S phases but are more susceptible to the Fas pathway.

Animals↗
PubMed1998

Entry and trafficking of granzyme B in target cells during granzyme B-perforin-mediated apoptosis.

In the widely accepted model of granule-mediated killing by cytotoxic lymphocytes, granzyme B entry into the target cell is facilitated by the pore forming molecule, perforin. Using indirect immunofluorescence and also direct visualization of fluorescein isothiocyanate (FITC)-conjugated granzyme B, we demonstrate internalization in the absence of perforin. Induction of the lytic pathway, however, required a second signal that was provided by perforin or adenovirus (Ad2). The combination of agents also resulted in a dramatic relocalization of the granzyme. Microinjection of granzyme B directly into the cytoplasm of target cells resulted in apoptosis without the necessity of a second stimulus. This suggested that the key event is the presence of granzyme B in the cytoplasm, and that when the enzyme is internalized by a target cell, it trafficks to an intracellular compartment and accumulates until release is stimulated by the addition of perforin. We found that the proteinase passed through rab5-positive vesicles and then accumulated within a novel compartment. On the basis of these results, we propose a new model for granzyme-perforin-induced target cell lysis in which granzyme B is subjected to trafficking events in the target cell that control and contribute to cell death.

Adenoviridae↗
PubMed1998

Evidence for the involvement of Fas ligand and perforin in the induction of vascular leak syndrome.

Endothelial cell injury resulting in vascular leak syndrome (VLS) is one of the most widely noted phenomenons in a variety of clinical diseases. In the current study we used IL-2-induced VLS as a model to investigate the role of cytolytic lymphocytes in the cytotoxicity of endothelial cells. Administration of IL-2 (75,000 U/mouse, three times a day for 3 days) into BL/6 wild-type mice triggered significant VLS in the lungs, liver, and spleen. Interestingly, perforin-knockout (KO) mice exhibited a marked decrease in IL-2-induced VLS in all three organs tested. Also, Fas ligand-defective (gld) mice and Fas-deficient (lpr) mice exhibited decreased VLS in the liver and spleen, but not in the lungs. The decreased VLS seen in perforin-KO, gld, and lpr mice was not due to any defect in lymphocyte migration or homing to various organs because histopathologic studies in these mice demonstrated significant and often greater perivascular infiltration of lymphocytes compared with the IL-2-treated wild-type mice. Ultrastructural studies of the lungs demonstrated significant damage to the endothelial cells in IL-2-treated wild-type mice and decreased damage in perforin-KO mice. IL-2 administration caused up-regulation of CD44 in all strains of mice tested and triggered increased LAK activity against an endothelial cell line in wild-type and gld mice, but not in perforin-KO mice. The current study demonstrates for the first time that perforin and Fas ligand may actively participate in endothelial cell injury and induction of VLS in a variety of organs.

Animals↗
PubMed1998

Age-related decline of perforin expression in human cytotoxic T lymphocytes and natural killer cells.

In this study a flow cytometric technique for detecting cytoplasmic perforin (P) has been used to quantify age-related changes in perforin expression in human peripheral blood lymphocytes (PBL). Proportions of P+ lymphocytes increased after birth, but declined rapidly after the age of 70 years. This was true for both T cells and CD16(+) and CD56(+) natural killer (NK) cells. Children showed in addition to high levels of perforin positive CD8(+) cells a much higher proportion of CD4(+)P+ cells than the other age groups. In elderly individuals there was also a highly significant reduction in mean levels of perforin per cell as compared with all other groups (P < .05 to .001). Adult women had consistently higher mean levels of perforin per cell than adult men for all P+ cell phenotypes. Functional tests clearly showed the deficiency in early spontaneous cytotoxic potential of PBL from elderly persons due to relative P deficiency, which can be corrected by stimulation of cytolytic cells with target cells and interleukin-2 (IL-2). The deficiency in cytolytic activity on the contact with target cells may have implications for antiviral and antitumor immunity in elderly persons.

Adult↗
PubMed2001

Evidence for perforin-like activity associated with earthworm leukocytes.

Earthworm (Eisenia fetida) coelomic fluid contains several leukocytes (coelomocytes): basophils, acidophils and neutrophils as well as chloragocytes. Small coelomocytes and coelomocyte lysate are cytotoxic for the tumor cell target K562. The expression of a lytic factor was investigated by immunocytochemistry using light and transmission electron microscopy. A rat-anti-mouse-perforin-mAb labeled mainly small coelomocytes (nearly 20%) as visualized by light microscopy. TEM analysis using immunogold showed a homogenous labeling in the cytoplasm of small coelomocytes. The highest number of immunogold particles was estimated in coelomocytes with many small cytoplasmic granules. Coelomocytes with large lysosomal granules were also labeled but less intensely. No antibody binding was observed for chloragocytes either in light or electron microscopy. This suggests that the perforin-like activity is associated with only one cell type and that chloragocytes are responsible for other lytic activities. MALDI-MS revealed calreticulin usually associated with perforin in mammalian cells that mediate lysis (e.g. NK, CTL). Together, results strongly suggest the presence of putative perforin in earthworms. This in turn supports the hypothesis that perforin is a conserved component important in immune defense during evolution.

Journal Article↗
PubMed1999

In situ detection of tumor infiltrating lymphocytes expressing perforin and fas ligand genes in human HCC.

AIM:To investigate the expression of perforin and fas-ligand (fas-L) of tumor infiltrating lymphocytes (TILs) in human hepatocellular carcinoma (HCC).METHODS:By in situ hybridization and immunohistochemistry, the perforin and fas-L gene expression of TILs was studied in 20 HCC cases.RESULTS: Positive expression of perforin and fas-L genes was detected in 16 HCC cases. One patient had expression of perforin and fas-L genes in the majority of TILs and survived 1.5 years after tumor resection without HCC relapse.This seems that the presence of a large number of activated T cells might be beneficial for the antitumor immunity. In other cases, less than 10% of TILs were able to express perforin and fas-L genes.CONCLUSION:Although there were a number of T cells in HCC, only few of them were immunoactive and able to kill tumor cells. It seems important to promote further proliferation of these activated T cells in vitro or in vivo.

Journal Article↗
PubMed2006

NK cells use NKG2D to recognize a mouse renal cancer (Renca), yet require intercellular adhesion molecule-1 expression on the tumor cells for optimal perforin-dependent effector function.

The NKG2D receptor on NK cells can recognize a variety of ligands on the tumor cell surface. Using a mouse renal cancer (Renca), we show that NKG2D recognition by NK cells was crucial for their ability to limit tumor metastases in vivo in both liver and lungs using perforin-dependent effector mechanisms. However, for the R331 cell line established from Renca, NKG2D recognition and perforin-dependent lysis played no role in controlling liver metastases. R331 cells were also more resistant to perforin-dependent lysis by NK cells in vitro. We therefore used these phenotypic differences between Renca and R331 to further investigate the crucial receptor:ligand interactions required for triggering lytic effector functions of NK cells. Reconstitution of R331 cells with ICAM-1, but not Rae-1gamma, restored NKG2D-mediated, perforin-dependent lysis. Interestingly, R331 cells were efficiently lysed by NK cells using death ligand-mediated apoptosis. This death ligand-mediated killing did not depend on NKG2D recognition of its ligands on tumor cells. This result suggests that the intracellular signaling in NK cells required for perforin and death ligand-mediated lysis of tumor target cell are quite distinct, and activation of both of these antitumor lytic effector functions of NK cells could improve therapeutic benefits for certain tumors.

Animals↗
PubMed1997

Impaired nonrestricted cytolytic activity in systemic lupus erythematosus: involvement of a pathway independent of Fas, tumor necrosis factor, and extracellular ATP that is associated with little detectable perforin.

OBJECTIVE: To determine the cytolytic effector pathway involved in impaired generation of nonrestricted cytolytic activity in systemic lupus erythematosus (SLE). METHODS: Peripheral blood mononuclear cells (PBMC) from normal subjects and SLE patients were stimulated in vitro with anti-CD3 monoclonal antibody (MAb) and interleukin-2 to promote the generation of nonrestricted cytolytic activity. On day 13 of culture, the PBMC were assayed for cytolytic activity against Fas-Daudi cells and Fas+ Jurkat cells. The effects on cytotoxicity of calcium chelation, antagonist anti-Fas MAb, tumor necrosis factor (TNF) alpha and beta, and ATP were measured. Intracellular perforin expression was determined by intracellular staining, and perforin messenger RNA levels were determined by quantitative competitive reverse transcription-polymerase chain reaction. RESULTS: We demonstrated the existence of a cytolytic pathway that is independent of Fas, TNF alpha, TNF beta, and ATP, but is dependent upon extracellular calcium. Despite its calcium dependence, this pathway is associated with low-to-undetectable levels of perforin. CONCLUSION: Impaired generation of nonrestricted cytolytic activity in SLE is likely due to decreased activity of this Fas-, TNF alpha-, TNF beta-, ATP-independent pathway associated with very low levels of perforin.

Adenosine Triphosphate↗
PubMed2003

Novel perforin mutation in a patient with hemophagocytic lymphohistiocytosis and CD45 abnormal splicing.

Hemophagocytic lymphohistiocytosis (HLH) composes a group of rare heterogenous disorders characterized by uncontrolled accumulation and infiltration of activated T lymphocytes and macrophages. Cytotoxic T and natural killer cell activity is significantly reduced or absent in these patients. Mutations in the important mediator of lymphocyte cytotoxicity perforin were identified in a number of HLH individuals. Here we report a novel missense mutation thr435met in the conserved Ca(2+) binding domain of perforin in a patient with HLH. Prediction of the 3-dimensional structure of the thr435met perforin mutant using comparative molecular modeling indicates that the protein's ability to bind Ca(2+), and therefore its cytolytic function, would be strongly compromised. In addition, this patient exhibited abnormal CD45 splicing caused by a C77G mutation in the gene encoding CD45 (PTPRC). Our findings suggest a combined role for perforin mutation and abnormal CD45 splicing as significant contributory factors in the pathogenesis of HLH.

Alternative Splicing↗
PubMed2000

Fas- and tumor necrosis factor receptor 1-dependent but not perforin-dependent pathways cause injury in livers infected with an adenovirus construct in mice.

Intravenous injection of type 5 adenovirus, deleted in the E1 and E3 regions, is shown to result in expression of viral antigens in the liver, initiating lymphocyte infiltration and liver injury. Following this infection, induction of Fas ligand (FasL), tumor necrosis factor alpha (TNF-alpha), and perforin mRNA are all demonstrable in the liver, pointing to a role of respective pathways in liver injury. Making use of mice in which the genes coding for Fas, FasL, TNF receptors (TNFRs), and perforin are inactivated, as well as recombinant proteins that inhibit Fas- and TNF-alpha-mediated apoptosis, it is shown that a functional perforin-mediated mechanism is not obligatory for cellular infiltration and progression of liver injury. In contrast functional Fas- and TNF-alpha-mediated mechanisms were found to be essential for liver injury to occur. Results are presented demonstrating that signaling through TNFR1, but not TNFR2, is involved in TNF-alpha-mediated liver damage. The conclusion is drawn that although perforin mRNA is induced in the virus-infected liver, Fas- and TNF-alpha-mediated mechanisms constitute the principal pathways by which the cell-mediated immune system causes acute liver injury.

Adenoviridae↗
PubMed1990

Transients of perforin pore formation observed by fluorescence microscopic single channel recording.

A new type of single channel recording is described. Large pores were generated in the membranes of resealed human erythrocyte ghosts by incubation with perforin (cytolysin). The flux of the polar fluorescent probe Lucifer Yellow was measured in single ghosts by the fluorescence microphotolysis (photobleaching) technique. The distribution of flux rates for ghosts treated with a limiting perforin concentration showed equidistantly spaced peaks suggesting that subpopulations of ghosts with 0, 1 and 2 pores were resolved. Furthermore, distributions obtained for very different perforin concentrations could be well simulated by using one common value for the flux rate of the single pore (k = 4.65 x 10(-3) s) and assuming a Poisson distribution of pores among ghosts. The flux rate of the single pore corresponds to a pore radius of approximately 50 A, a value which is much smaller than that obtained previously by electron microscopic studies but which agrees well with recent electrical single channel recordings. Mature perforin pores were observed to be very stable. No closing events were detected at a time resolution of 0.2 s for a wide range of temperatures and Ca2+ concentrations. However, the formation of new pores was an unexpectedly slow process. Fluorescence microscopic single channel recording as introduced by this study is applicable to a variety of cellular systems and fluorescent probes and thus may complement the information obtainable by electrical single channel recording of anorganic ion fluxes.

Erythrocyte Membrane↗
PubMed1994

Macrophage precursor cells produce perforin and perform Yac-1 lytic activity in response to stimulation with interleukin-2.

Macrophage precursor cells, derived from mouse bone marrow culture with granulocyte-macrophage colony-stimulating factor or colony-stimulating factor 1 (CSF-1) as growth factor and interleukin-2 (IL-2) as stimulating factor, were activated by IL-2 to exert strong cytolytic activity against Yac-1 cells. In response to IL-2 stimulation these bone marrow macrophage precursor cells produced perforin as lytic molecules. The purity of the precursor cells for the study was proved as homogeneous positivity for Mac-1, NK-1.1 and negativity for Lyt 1 and 2. The cells express CSF-1 receptors on their surface, are able to proliferate and differentiate into typical macrophages when stimulated with CSF-1, and are therefore members of the macrophage lineage. Perforin transcripts were identified by Northern blot analysis of IL-2-treated macrophage precursor cells, and the presence of perforin protein in the cytoplasmic granules was demonstrated by immunohistochemical staining using a monoclonal antiperforin antibody. In addition, the biological activity of the perforin contained in the macrophage precursor's granules could be documented as calcium-dependent lytic activity using Yac-1 and sheep red blood cells as targets. The results presented in this paper imply the existence of a bipotent precursor cell, which can mature into a typical macrophage if CSF-1 or phorbol 12-myristate 13-acetate is supplied as differentiation stimulating factor but develops into an NK/LAK cell when early activation with IL-2 is provided.

Animals↗
PubMed2002

Biology and treatment of familial hemophagocytic lymphohistiocytosis: importance of perforin in lymphocyte-mediated cytotoxicity and triggering of apoptosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is, without treatment, an invariably fatal disease of infancy and early childhood characterized by fever, hepatosplenomegaly, pancytopenia, and a widespread accumulation of T-lymphocytes and macrophages. During recent years, the diagnosis and the survival as well as the understanding of the disease have improved dramatically. Recent studies suggest that FHL is caused by impaired lymphocyte-mediated cytotoxicity and defective triggering of apoptosis, and that the symptoms are mediated by a pro-inflammatory hypercytokinemia. Moreover, specific genetic alterations, mutations in the perforin gene, have been revealed in FHL patients. Perforin, which normally is secreted from cytotoxic T-lymphocytes and natural killer (NK) cells upon conjugation between effector and target cells, is able to insert into the membrane of the target cell. It there polymerizes to form a cell death-inducing pore through which toxic granzymes may enter the cell and trigger apoptosis. The establishment of perforin deficiency as a cause of the rapidly fatal disease FHL has demonstrated the essential role of perforin in human immune homeostasis.

Apoptosis↗
PubMed1997

The role of phorbol ester-sensitive protein kinase C isoforms in lymphokine-activated killer cell-mediated cytotoxicity: dissociation between perforin-dependent and Fas-dependent cytotoxicity.

Treatment of lymphokine-activated killer (LAK) cells with phorbol ester (PMA) caused the downmodulation of LAK activity concomitantly with the inhibition of serine esterase (SE) release, which has been shown as a marker for perforin-dependent cell-mediated cytotoxicity. The reduction of perforin-dependent LAK activity by PMA-treatment appeared to be due to the disappearance of PMA-sensitive protein kinase C (PKC) isoforms such as PKC alpha, gamma, epsilon, theta. In contrast, Fas-mediated LAK activity was refractory against PMA-induced downregulation. Treatment of LAK cells with PMA caused a disappearance of cytotoxicity against Fas L5178Y tumor cells, while cytotoxicity against Fas+ transfectants was not affected by PMA treatment. Moreover, Fas-mediated LAK activity of perforin-knockout mice was not inhibited by PMA treatment. These results clearly demonstrated that Fas-mediated cytotoxicity could be dissociated from perforin-mediated cytotoxicity by their different requirement of PMA-sensitive PKC isoforms.

Animals↗
PubMed1995

IL-12 synergizes with IL-2 to induce lymphokine-activated cytotoxicity and perforin and granzyme gene expression in fresh human NK cells.

NK-mediated cytotoxicity is regulated by a variety of cytokines and is thought to involve perforin and granzymes. The effects of IL-2 and IL-12 on the expression and activation of cytolysis were examined in freshly isolated human NK cells. A dose-dependent increase in cytolysis of the NK-sensitive target cell, K562, and the NK-insensitive but lymphokine-activated killer (LAK) cell-sensitive target, UCLA-SO-M14, was observed after short term culture of purified human NK cells in either IL-2 or IL-12. Moreover, the two cytokines often synergized to produce augmented lytic activity. A suboptimal dose of IL-2 (60 IU/ml) combined with IL-12 (2 U/ml) could induce lytic activity equal to twice the additive effect of each cytokine alone. Northern analyses revealed time-dependent increases in mRNAs encoding for perforin and granzymes A and B following treatment with IL-2 alone or IL-2 plus IL-12. IL-2 and IL-12 also synergized for the induction of granzyme mRNAs, in that treatment with both cytokines increased mRNA levels approximately 50% above the sum of each cytokine alone, as quantitated by phosphorimage analysis, and normalized to GAPDH gene expression. However, the synergy between IL-2 and IL-12 for the induction of mRNA was less dramatic than for lytic activity. Results of experiments in which cytokine-treated cells were pulsed with actinomycin D indicated that the increased granzyme and perforin gene mRNA levels in response to IL-2, IL-12, or the combination were not due to increased transcript stability. The data suggest that low doses of IL-2 and IL-12 synergize to augment NK- and induce LAK-mediated cytotoxicity and that this increase is associated with enhanced transcription of perforin and granzyme genes in a synergistic fashion.

Cells, Cultured↗
PubMed2002

Interferon-gamma secretion and perforin expression are impaired in CD8+ T lymphocytes from patients with undifferentiated carcinoma of nasopharyngeal type.

An efficent antitumor and antiviral cellular immune response requires optimal interferon-gamma (IFN-gamma) secretion and perforin expression in CD8(+) T cells. The aim of this study was to define whether CD4(+) and CD8(+) T cells from patients with undifferentiated carcinoma of nasopharyngeal type (UCNT), a tumor regularly associated with the Epstein-Barr virus (EBV), have abnormal phenotype profiles, cytokine production, perforin and CD3-zeta expressions. Our data showed that CD4 and CD8 subset distribution was not grossly altered in the peripheral blood of UCNT patients, while tumor biopsies contained an increased proportion of CD8(+) T cells. The analysis of the CD4(+) subset showed a defect in interleukin-2 (IL-2) production and a moderate increase of IL-10 production, a situation consistent with a Th1/Th2 imbalance. We have also demonstrated that CD8(+) lymphocytes from UCNT patients had a marked impairment of IFN-gamma secretion and perforin expression. This impairment was not related to the presence of detectable EBV DNA in the plasma. In UCNT patients, the blockade of the perforin pathway and of IFN-gamma production may constitute important mechanisms for immune escape by the tumor and for impaired control of EBV replication.

Adult↗
PubMed2004

Paclitaxel probably enhances cytotoxicity of natural killer cells against breast carcinoma cells by increasing perforin production.

Paclitaxel, a semisynthetic taxane, is one of the most active chemotherapeutic agents for the treatment of patients with breast cancer. We focused on the effect of paclitaxel on the cytotoxicity of natural killer (NK) cells. NK cells were purified by negative selection with magnetic beads from peripheral blood mononuclear cells of healthy volunteers. A human breast carcinoma cell line BT-474 and an NK cell-sensitive erythroleukemia cell line K562 were used as targets. Cytotoxicity of NK cells was determined by 51Cr-release assay with labeled target cells. Paclitaxel (1-100 nM) did not affect cellular viability, and significantly enhanced cytotoxicity of NK cells in a dose-dependent manner. Although paclitaxel did not affect Fas-ligand expression of NK cells, paclitaxel induced mRNA and protein production of perforin, an effector molecule in NK cell-mediated cytotoxicity. Concanamycin A, a potent inhibitor of the perforin-mediated cytotoxic pathway, inhibited paclitaxel-dependent NK cell-mediated cytotoxicity. Furthermore, paclitaxel induced activation of nuclear factor kappa B (NF-kappa B) in NK cells. NF-kappa B inhibitor pyrrolidine dithiocarbamate significantly suppressed both paclitaxel-induced perforin expression and NK cell cytotoxicity. Our results show for the first time that paclitaxel enhances in vitro cytotoxicity of human NK cells. Moreover, our results suggest a significant association between enhanced NK cell cytotoxicity, increased perforin production, and NF-kappa B activation.

Adenocarcinoma↗