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PubMed2006

Differential expression of perforin in cytotoxic lymphocyte in HIV/AIDS patients of China.

Cytotoxic lymphocytes are critical in the control of HIV replication, it has been shown that perforin is the key effector of killing machinery for CTLs and NK cells, so we investigated the circulating levels of perforin in CD8+ T cells and NK cells by flow cytometry intracellular stain in Chinese HIV infected individuals, its association with disease progression was analyzed. Our results showed that NK cells express perforin more efficiently than CD8+ T cells, CD8+ T cells expressed perforin higher than that of healthy controls, but NK cells expressed lower perforin than that of healthy controls, both were not correlated with disease progression. but significantly associated with their numbers, anti-retrovirus therapy had no evident effects on peforin expression in CD8+ T cells, but enhanced perfrin expression in NK cells, perforin expression in CD8+ T cells and CD16+ NK cells correlate with CD4+ T cell counts significantly in HAART-treated group. Therefore, different mechanisms may be involved in regulating peripheral perforin expression in different cell types.

Adult↗
PubMed1993

Perforin expression localizing cytotoxic lymphocytes in the intimas of coronary arteries with transplant-related accelerated arteriosclerosis.

Accelerated arteriosclerosis is the major long-term complication of cardiac transplantation. It has been demonstrated recently that accelerated arteriosclerosis is caused, in part, by rejection-related, cell-mediated immunity. However, the role of cytotoxic T lymphocytes in this process is a subject of controversy. Perforin is a specific marker of functionally active cytotoxic lymphocytes because it is a functional component of the cytotoxic granules of these cells. We examined 11 coronary arteries from seven autopsied and four retransplanted heart transplant recipients for the presence of perforin-containing lymphocytes. Immunohistochemical stains for perforin were performed using a monoclonal antibody against human perforin. Eight of the 11 coronary arteries examined were found to contain perforin-positive cells. These perforin-positive cells were present in subendothelial spaces of the coronary arteries, and the staining seen was cytoplasmic and granular. The granules often were polarized to the endothelial surface. Furthermore, the cells identified were usually in close proximity to, or in direct contact with, coronary artery endothelial cells. These results suggest that cell-mediated endothelial injury by perforin-positive cytotoxic lymphocytes may contribute to the development of accelerated arteriosclerosis in heart transplant recipients.

Adolescent↗
PubMedDate not supplied

Target cell-induced perforin mRNA turnover in NK3.3 cells is mediated by multiple elements within the mRNA coding region.

We have previously shown that in cytolytic cells exposed to sensitive targets the mRNA of the cytolytic protein perforin undergoes rapid downregulation. We now demonstrate that perforin message undergoes accelerated turnover in NK3.3 cells exposed to sensitive TC. This inducible mRNA decay phenomenon is specific for cytolytic protein messages, as levels of the constitutive message beta-actin are unchanged. This TC-induced perforin mRNA turnover cannot be attributed to a blockage of RNA synthesis, or to a rapid half-life (t 1/2). To determine the region(s) within the perforin transcript responsible for governing this TC-mediated turnover event, various segments of the perforin cDNA were cloned and inserted into the 3' UTR of rabbit beta globin (RG). Constructs containing perforin coding region cDNA, but not 3' UTR cDNA, mediated TC-induced mRNA turnover. These data indicate that multiple elements governing perforin mRNA stability reside within the coding region, a novel type of mRNA regulation not previously described.

Animals↗
PubMed2005

Perforin improves the morphogenesis of mouse placenta disturbed by IL-2 treatment.

The pore-forming protein (perforin) produced by lymphocytes can induce apoptosis in target cells. In mouse placenta, although a large amount of perforin is produced by the uterine natural killer (uNK) cells, its role in the reproductive process is still not clear. Since the cytotoxicity of uNK cells can be enhanced by interleukin (IL)-2, we studied the role of perforin in the placenta of wild-type and perforin-knockout mice treated with IL-2 during days 10-14 of pregnancy. Immunohistochemistry of the wild-type mice showed that the perforin was positive in the membrane of trophoblast glycogen cells as well as the cytoplasm of uNK cells, and there was an increase in the expression level following IL-2 treatment as revealed by RT-PCR analysis, although no change was identified in fertility. In the IL-2-treated perforin-knockout mice, however, the number of live fetuses was decreased, accompanied by an increase in the weight of placentae. Examination of these placentae showed an abnormally enlarged junctional zone, occupied by a large number of the trophoblast glycogen cells and significantly few of the apoptotic cells. These findings indicate that perforin can contribute to a successful pregnancy by inhibiting the excessive growth of the junctional zone induced by IL-2.

Animals↗
PubMed2004

Tributyltin exposure causes decreased granzyme B and perforin levels in human natural killer cells.

Natural Killer (NK) cells are a subset of lymphocytes that are capable of killing tumor cells, virally infected cells and antibody coated cells. Tributyltin (TBT) is a toxic chemical used for various industrial purposes such as: slime control in paper mills, disinfection of circulating industrial cooling waters, anti-fouling agents in shower curtains and the preservation of wood. TBT can be found in edible items such as dairy products and fish. This study investigates the mechanism by which TBT exposure decreases the immune function of human NK cells, in vitro. Cytotoxic function, the expression of the cytotoxic proteins (granzyme B and perforin), and cAMP response element binding protein (CREB) phosphorylation were examined. NK cells exposed to 300 nM TBT for 1 h showed no significant decrease in cytotoxic function, levels of granzyme B and perforin, or phosphorylation of CREB. However, mRNA levels for the cytotoxic proteins were significantly decreased. A 24 h exposure to 200 nM TBT caused significant decreases in cytotoxic function, levels of granzyme B and perforin, and levels of granzyme B and perforin mRNA. When NK cells were exposed to 300 nM TBT for 1h followed by a 24 h period in TBT-free media, again there were significant decreases in NK cell cytotoxic function, levels of granzyme B and perforin and their mRNA. A 1h exposure to 300 nM TBT followed by a 48 h period in TBT-free media showed similar changes in cytotoxic function and levels of granzyme B and perforin as seen after 24 h in TBT-free media. Additionally, both of these exposures showed significant decreases in phosphorylation of CREB. These results indicate that TBT exposures can disrupt the transcription of granzyme B and perforin and that this disruption cannot be entirely accounted for by a decrease in phosphorylated CREB (phosphoCREB) levels.

Adult↗
PubMed1998

Consequences of Fas-ligand and perforin expression by colon T cells in a mouse model of inflammatory bowel disease.

BACKGROUND & AIMS: We describe a type of colitis that develops after transplantation of nonallogeneic wt bone marrow cells into T cell- and natural killer cell-deficient Tg26 mice (BM-->Tg26). In these animals, severe wasting and inflammation of the colon correlates with the expansion of mucosal T lymphocytes that displays cytotoxic activity. The aims of this study were to determine the relative contribution of perforin and Fas ligand (Fas-L) expression to the cytotoxic action of these T cells and to examine the influence of each pathway in this model of colitis. METHODS: Colonic T cells were tested for their ability to mediate Fas- and perforin-dependent killing in redirected cytotoxicity assays. Bone marrow cells from donor mice lacking either Fas-L (gld mice) or perforin (PFPnull mice) or both molecules were used to reconstitute Tg26 mice. RESULTS: Colon cytotoxic T lymphocyte displayed both Fas- and perforin-dependent killing. Deficiency in perforin, but not Fas-L, resulted in reduced incidence of wasting and, to a lesser extent, severe colitis in BM-->Tg26 animals. CONCLUSIONS: Colon T cells from BM-->Tg26 mice express both perforin and Fas-L. Although neither pathway is critical in the development of colitis, perforin does have a measurable influence on disease in the BM-->Tg26 colitis model.

Animals↗
PubMed2003

Local immune responses to influenza virus infection in mice with a targeted disruption of perforin gene.

The role of perforin in the local defense mechanisms against influenza virus infection was investigated. Mice deficient in the perforin gene (perforin(-/-)) were more susceptible to influenza virus infection than the ordinary wild-type C57BL/6 mice, showing an increased mortality with elevated virus growth and prolonged virus shedding. The lung parenchyma cells of perforin(-/-) mice showed no cytolytic activities of natural killer cells or virus-specific cytotoxic T lymphocytes in vitro, although the local antibody production system in the respiratory tract functioned well. In perforin(-/-) mice, the appearance of apoptotic degeneration in virally infected lung cells was delayed. This might cause cellular infiltration, including CD4, CD8, and CD19 positive cells, in the lung, peaking at day 8 after infection and maintaining a high level for a longer period. In the virus-induced local cytokine production, interferon-gamma (IFN-gamma) was prominent. The adoptive transfer of immune-competent spleen cells from wild-type C57BL/6 mice achieved a complete protection of perforin(-/-) mice against lethal challenge infection. These results suggest that perforin plays a crucial role in the host defense system against influenza virus infection, especially in its early stage, by inducing apoptosis of virus-infected cells.

Adoptive Transfer↗
PubMed2004

In vivo rejection of tumor cells dependent on CD8 cells that kill independently of perforin and FasL.

Perforin/granzyme B- and Fas/FasL-mediated killing pathways are the main effector mechanisms of CTL and NK cells in antitumor immune responses. In this study, we investigated the relative role of these two lytic mechanisms in protection of the host from tumor progression, as well as spontaneous metastasis, using the D122 Lewis lung carcinoma and its gene-modified cells. Utilizing perforin knockout mice (B6-PKO) and Fas and FasL mutant (B6-MRL and B6-Smn) mice, we found that perforin expression in the host plays a crucial function in the prevention of metastasis. However, local tumor rejection of an H-2K(b) and B7-1 transfectant, 39.5-B7 cells, was not dependent either on perforin or Fas/FasL expression in vivo. In addition, CTL lysis of 39.5-B7 cells was independent of perforin and Fas/FasL interactions in 18-hour in vitro assays. We also confirmed that CD8 T-cells were responsible for rejecting 39.5-B7 local tumors, yet cytokines, TNF-alpha and gammaIFN were not involved in tumor rejection in vivo. Furthermore, blocking assays using caspase inhibitors (zVAD-fmk, zLETD-fmk and zLEHD-fmk) showed that, whereas caspase activation was partially required to induce 39.5-B7 lysis mediated by the perforin-dependent pathway, 39.5-B7 lysis by CTLs through the perforin-independent mechanism required caspase activation. Thus, these results suggested that perforin, Fas/FasL, gammaIFN and TNF-alpha independent lytic mechanisms, mediated by CD8 T cells, have a crucial role in rejection of 39.5-B7 cells in vivo. Caspase activation is a pre requisite for apoptosis of targets by CTLs.

Animals↗
PubMed1992

Perforin gene expression in T lymphocytes correlates with disease activity in immunoglobulin A nephropathy.

1. We studied perforin gene expression in T lymphocytes obtained from 26 patients with IgA nephropathy and from 15 healthy age-matched control subjects. 2. The majority of patients with IgA nephropathy (96%) had elevated perforin mRNA expression, whereas no perforin mRNA expression was detected in the T lymphocytes of normal control subjects. 3. A positive correlation was noted between perforin mRNA expression and urinary protein excretion. 4. Perforin mRNA expression correlated also with the histopathology in the renal tissue of patients with IgA nephropathy. 5. Sixty per cent of patients with grade III or IV histopathology had high perforin mRNA expression in T lymphocytes [more than (++)]. 6. These studies suggest that disregulation of perforin gene expression in T lymphocytes may be associated with the progression of IgA nephropathy and could be used as an indicator of disease activity.

Adolescent↗
PubMed2002

Epstein-Barr virus-specific T-cell cytotoxicity is mediated through the perforin pathway in patients with lymphoproliferative disorders after allogeneic bone marrow transplantation.

The in vivo cytotoxic mechanism of Epstein--Barr virus (EBV)-specific cytotoxic lymphocytes was examined in a patient who suffered with EBV-associated lymphoproliferative disease (LPD) after bone marrow transplantation (BMT). His peripheral CD8+ T-cell count was significantly increased and >70% of these cells were EBV-specific by fluorescence-activated cell sorter (FACS) analysis for interferon-gamma production. Intracellular perforin expression was markedly increased in CD8+ T cells by FACS analysis. The lymphocytes from this patient had cytotoxic activity against autologous EBV+ lymphoblastoid cell lines which were completely inhibited by concanamycin A, an inhibitor of perforin, and a anti-human leucocyte antigen (HLA)-class I monoclonal antibody. These results suggest that the cytotoxicity was mediated by the perforin, in an HLA-class I-restricted manner. We performed serial intracellular perforin analyses in another patient who also showed endogenous expansion of EBV-specific CD8+ T cells that coincided with an increased EBV-DNA load. Perforin expression in the CD8+ and CD4+ T cells paralleled the EBV-specific CD8+ T cells and EBV-DNA load, which also suggests that perforin mediates EBV-specific cytolysis in vivo and is responsible for effective immunosurveillance against EBV reactivation after BMT. Evaluation of host immunity against EBV by determining perforin expression in lymphocytes and EBV-specific lymphocytes along with quantification of EBV-DNA may be useful for predicting the clinical course of patients with EBV-associated LPD after BMT.

Biomarkers↗
PubMed2003

Characteristic perforin gene mutations of haemophagocytic lymphohistiocytosis patients in Japan.

Perforin gene (PRF1) mutations appear to occur in about 30% of patients with haemophagocytic lymphohistiocytosis (HLH). We tested perforin expression and gene mutations in 14 HLH patients and six patients with Epstein-Barr virus-associated HLH (EBV-HLH) in Japan. Five of the 14 HLH patients had perforin abnormalities. The presence of PRF1 genetic abnormality correlated well with the lack of perforin expression as determined by flow cytometry. Sequencing showed that four patients had a compound heterozygous mutation while the fifth patient had a homozygous mutation. Three of the mutations we detected were novel. In contrast, none of the six EBV-HLH patients showed perforin abnormalities. Our data, combined with the PRF1 mutations in three previously reported Japanese patients, suggest that the 1090-1091delCT and 207delC mutations of the perforin gene are frequently present in Japanese HLH patients (62.5% and 37.5% respectively). Examination of the geographical origins of the ancestors in the perforin-mutant HLH patients revealed that they mostly came from the Western part of Japan, suggesting that the present-day cases may largely derive from a common ancestor.

Adolescent↗
PubMed1999

Perforin and Fas/Fas ligand-mediated cytotoxicity in acute and chronic woodchuck viral hepatitis.

The Fas ligand (FasL)/Fas and the perforin-granzyme cytotoxic pathways presumably play a central role in the development of hepatocellular injury in viral hepatitis. To recognize the potential contribution of FasL and perforin-based cell killing in hepadnaviral infection, we adopted a cytotoxic assay using murine Fas+ P815 and human Fas- K562 cells as targets. Freshly isolated peripheral blood mononuclear cells (PBMC) from woodchucks with newly acquired woodchuck hepatitis virus (WHV) infection (n = 6), with chronic WHV hepatitis (n = 9), and from healthy animals (n = 11) were used as effector cells. We have found that woodchuck lymphoid cells kill cell targets via both the FasL/Fas and the perforin death pathways. The contribution of Fas-dependent cytolysis was ascertained in blocking experiments with anti-Fas antibody and by incubation of PBMC with cyclohexamide to prevent de novo synthesis of FasL. The involvement of the perforin pathway was confirmed by treatment of K562 cells with colchicine to inhibit the microtubule-dependent perforin release. Comparative analysis showed that peripheral lymphoid cells from acute WHV hepatitis, but not those from chronic WHV infection, are more cytotoxic and that this increase seems to be entirely due to activation of perforin-mediated killing. The data indicate that acute infection in woodchucks is associated with the augmented capacity of lymphoid cells to elicit perforin-dependent killing, but in chronic infection, independent of the severity of liver disease and duration of chronicity, these cells have the same or lower cytotoxic potential as PBMC from healthy controls. These findings suggest a role for non-specific cellular immunity, presumably natural killer (NK) cells, in the control of early WHV infection and in the progression of chronic hepatitis.

Acute Disease↗
PubMed2000

[Increase in perforin-positive peripheral blood lymphocytes in extrinsic and intrinsic asthma].

The cause of asthma which has been linked to a chronic, T-cell-mediated bronchial inflammation, remains unclear. A number of other T-lymphocyte-mediated, chronic inflammatory disorders have been associated with autoimmunity and there are data indicating that autoimmune phenomena might also be present in asthma. Expression of perforin, a cytotoxic molecule produced by lymphocytes, has been implicated in the pathogenesis of autoimmune disease. We therefore tested the hypothesis that allergic and intrinsic asthma might be associated with an increase in lymphocytes producing perforin by comparing the expression of intracellular perforin in peripheral blood lymphocytes of patients with extrinsic asthma (n = 13), intrinsic asthma (n = 7), and healthy (control subjects (n = 18). Lymphocytes were identified using flow cytometry and subdivided into CD3(+), CD4(+), CD8(+), CD16(+), and CD56(+) subpopulations after staining with appropriate monoclonal antibodies. The percentage of perforin-positive total lymphocytes as significantly elevated in patients with allergic as well as intrinsic asthma when compared with normal control subjects. Analysis of lymphocyte subpopulations also revealed a significant increase in the percentage of CD3(+), CD4(+), CD8(+), and CD56(+) cells expressing perforin in allergic asthma and a significant increase in the percentage of CD4(+), and CD56(+) cells in intrinsic asthma when compare with healthy control subjects. Perforin expression in CD4(+) cells in intrinsic asthma was also significantly elevated compared with allergic asthma. We conclude that allergic and intrinsic asthma is associated with increased expression of perforin in T-lymphocyte subsets.

Adult↗
PubMed1997

Reduced incidence and delayed onset of diabetes in perforin-deficient nonobese diabetic mice.

To investigate the role of T cell-mediated, perforin-dependent cytotoxicity in autoimmune diabetes, perforin-deficient mice were backcrossed with the nonobese diabetes mouse strain. It was found that the incidence of spontaneous diabetes over a 1 yr period was reduced from 77% in perforin +/+ control to 16% in perforin-deficient mice. Also, the disease onset was markedly delayed (median onset of 39.5 versus 19 wk) in the latter. Insulitis with infiltration of CD4(+) and CD8(+) T cells occurred similarly in both groups of animals. Lower incidence and delayed disease onset were also evident in perforin-deficient mice when diabetes was induced by cyclophosphamide injection. Thus, perforin-dependent cytotoxicity is a crucial effector mechanism for beta cell elimination by cytotoxic T cells in autoimmune diabetes. However, in the absence of perforin chronic inflammation of the islets can lead to diabetogenic beta cell loss by less efficient secondary effector mechanisms.

Animals↗
PubMed2001

Spectrum of perforin gene mutations in familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive disease of early childhood characterized by nonmalignant accumulation and multivisceral infiltration of activated T lymphocytes and histiocytes (macrophages). Cytotoxic T and natural killer (NK) cell activity is markedly reduced or absent in these patients, and mutations in a lytic granule constituent, perforin, were recently identified in a number of FHL individuals. Here, we report a comprehensive survey of 34 additional patients with FHL for mutations in the coding region of the perforin gene and the relative frequency of perforin mutations in FHL. Perforin mutations were identified in 7 of the 34 families investigated. Six children were homozygous for the mutations, and one patient was a compound heterozygote. Four novel mutations were detected: one nonsense, two missense, and one deletion of one amino acid. In four families, a previously reported mutation at codon 374, causing a premature stop codon, was identified, and, therefore, this is the most common perforin mutation identified so far in FHL patients. We found perforin mutations in 20% of all FHL patients investigated (7/34), with a somewhat higher prevalence, approximately 30% (6/20), in children whose parents originated from Turkey. No other correlation between the type of mutation and the phenotype of the patients was evident from the present study. Our combined results from mutational analysis of 34 families and linkage analysis of a subset of consanguineous families indicate that perforin mutations account for 20%-40% of the FHL cases and the FHL 1 locus on chromosome 9 for approximately 10%, whereas the major part of the FHL cases are caused by mutations in not-yet-identified genes.

Amino Acid Substitution↗
PubMed1994

Effects of purified perforin and granzyme A from cytotoxic T lymphocytes on guinea pig ventricular myocytes.

OBJECTIVE: Involvement of cytotoxic T lymphocytes (CTL) in heart transplant rejection as well as in viral myocarditis is well established, but the precise mechanisms whereby infiltrating CTL damage the myocardium are unknown. The aim of the study was to investigate how CTL derived perforin, the serine protease granzyme A, and the combination of both, damage guinea pig ventricular myocytes. METHODS: Action potentials and membrane currents were recorded by means of the whole cell configuration from guinea pig ventricular myocytes. RESULTS: Resembling the effects of CTL derived lytic granules, perforin caused gradual myocyte shortening and contracture, leading to complete loss of the rod shaped morphology and to cell destruction. These changes were preceded by shortening of action potential duration and reduction of resting potential and action potential amplitude, followed by complete inexcitability. Granzyme A alone was ineffective, but accelerated the deleterious effects of perforin on the morphological and electrophysiological properties of myocytes. The effects of perforin were further evaluated by measuring membrane currents by means of the whole cell voltage clamp. Perforin induced discrete changes in membrane current, reminiscent of single ion channels, with large conductance and open time of up to several seconds. Linear regression analysis of the channel I-V relations resulted in a conductance of 890 pS and a reversal potential of -7.6 mV. These results suggest that perforin induces large non-selective channels, which can account for most of the observed adverse effects. CONCLUSIONS: As CTL participate in the immunological rejection of the transplanted heart, it is conceivable, but remains to be shown, that part of this damage is inflicted by perforin containing lytic granules.

Action Potentials↗
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↗
PubMed1991

Expression of perforin by natural killer cells within first trimester endometrium in humans.

The lymphocyte pore-forming protein (PFP)--perforin, also named cytolysin--is a potent mediator of cytotoxicity found in the granules of cytotoxic T lymphocytes and natural killer (NK) cells. Granulated metrial gland (GMG) cells found in the pregnant mouse uterus express perforin and are thought to be highly activated cytolytic lymphocytes related to NK cells. Their role in pregnancy is unknown. Human endometrial granulocytes (EGs) are phenotypically similar to murine GMG cells and, like them, express NK cell markers. However, up to now it was not known whether EGs also express perforin. By means of immunohistochemical analysis, using antisera specific for perforin and monoclonal antibodies to CD56 (NKH-1), CD2 (T11), CD3 (Leu-4), CD4 (Leu-3a), and CD8 (OKT-8), we demonstrated that perforin is present in EGs in the decidualized endometrial stroma and decidual tissue of first-trimester gestational endometrium. In fact, double immunohistochemical labeling demonstrated the co-expression of perforin and NKH-1. This population also expressed some T-cell surface antigens (Leu-4 and T11), but not Leu-3a or OKT-8. Chorionic villi, in contrast, lack perforin+ cells. The presence of a potent cytolytic mediator in NK-like cells in both murine and human pregnant uterus raises the issue of the function of such cells in pregnancy.

Adult↗