Perforin expression in human peripheral blood mononuclear cells. Definition of an IL-2-independent pathway of perforin induction in CD8+ T cells.

Perforin gene expression upon in vitro stimulation was studied at the mRNA level in normal human PBMC and in subpopulations. Freshly isolated PBMC express low levels of perforin mRNA. Increased perforin expression is rapidly induced by the calcium ionophore A23187 and by rIL-2. Phorbolesters (PMA), by comparison, are poor inducers of perforin RNA. Perforin induction by Ca-ionophore, unlike granzyme 2 and IL-2 induction, did not synergize with phorbolesters in PBMC or in purified T cells. Instead, perforin mRNA induction by A23187 in purified T cells requires the presence of adherent cells. Ca-ionophore plus adherent cell-induced perforin occurred in CD8+ T cells and was abolished by depletion of CD8+ T cells but not by depletion of CD4+ T cells. Adherent cells alone did not express perforin under any condition. Perforin mRNA induction by both A23187 and by rIL-2 is independent of de novo protein synthesis. The half-life of perforin mRNA induced by either stimulus is approximately 100 min. Cyclosporin A completely abrogates perforin induction by A23187 but only slightly inhibits the effect of rIL-2 on perforin mRNA expression. These data show that A23187 activates perforin gene expression in CD8+ cells by an IL-2-independent pathway and that the molecular mechanism of perforin expression may be different from the one induced by IL-2. Granzyme 2 (human leukocyte protease-HLP, homologous to murine granzyme B) mRNA expression was studied in comparison to perforin. Granzyme 2 in contrast to perforin responds to the synergistic action of phorbolester and Ca-ionophore in PBMC. In addition, the kinetics of the induction of granzyme and perforin mRNA, by various signals are different. Our data suggest that situations in vivo may exist that allow perforin expression in CD8+ cells in the absence of cytokines by a combination of Ca signals and accessory receptor ligation. The same signals may not be sufficient for granzyme 2 expression in any T cell subpopulation.     

Apoptosis and apoptosis related proteins in chronic viral liver disease

Background: Apoptosis may be an important mechanism of hepatocyte death in chronic viral liver disease. Methods: We studied apoptosis in liver biopsies from 30 patients with chronic viral hepatitis and 8 patients with viral cirrhosis by the TUNEL method. 12 cases of non-alcoholic steatohepatitis and 12 cases of primary biliary cirrhosis were used as non-viral disease controls. Immunohistochemical expression of p53, p21/waf1, bcl-2 and mdm-2 proteins was also studied in the same patients. Results: A statistically significant increase of apoptotic liver cells was found in severe chronic viral hepatitis (5.3 ± 0.3%), cirrhosis (3.4 ± 0.5%) and PBC (4.4 ± 0.4%) cases compared to patients with non-alcoholic steatohepatitis (0.8 ± 0.3%). The expression of p53 protein was increased in the cases of viral cirrhosis and in chronic severe viral hepatitis whereas in the cases of chronic mild hepatitis, PBC and non-alcoholic steatohepatitis we found no expression of p53. P21/waf1 expression was increased in severe chronic hepatitis, cirrhosis and PBC cases compared to mild hepatitis and non-alcoholic steatohepatitis cases. However no induction of mdm-2 was observed in the subgroups of chronic liver disease. Bcl-2 was expressed only in epithelium of bile ducts and mononuclear cells of the portal tracts and liver lobules. A weaker Bcl-2 expression was noted in the epithelium of bile ducts of 7/12 PBC cases. Conclusion: Our results provide evidence of increased apoptosis in severe chronic viral liver disease, suggesting that apoptotic cell death might be involved in the pathogenesis of hepatocellular damage of viral hepatitis and cirrhosis. Furthermore we analysed part of the apoptotic pathways implicated in the above process and found an increased expression of p21/waf1, probably p53 mediated, without overexpression of the apoptosis inhibiting bcl-2 and mdm-2 proteins. By contrast p21/waf1 overexpression in PBC seems to be propagated by a p53 independent mechanism.     

Abundant expression of granzyme A, but not perforin, in granules of CD8+ T cells in GALT: implications for immune control of HIV-1 infection

Because GALT is a major portal of entry for HIV-1 and reservoir for viral replication, we hypothesized that an ineffective cellular immune response in intestinal mucosa might partially explain the failure of immune control in AIDS. In this study, we demonstrate that the vast majority of CD8+ T cells in rectal tissue, including HIV-1-specific cells, fail to express the cytolytic protein, perforin. However, rectal CD8+ T cells do express granzyme A, and are also capable of releasing IFN-gamma upon stimulation with cognate peptide. Confocal microscopy showed that granzyme A was located in intracellular granules in the absence of perforin. The majority of rectal CD8+ T cells exhibit an effector memory phenotype, expressing CD45RO but not CCR7. Quantitative real-time PCR analysis demonstrated that perforin RNA is expressed in rectal CD8+ T cells from healthy and HIV-1-positive individuals. In HIV-1-positive individuals, similar amounts of perforin RNA were detected in CD8+ T cells from rectal tissue and PBMC, despite a relative absence of perforin protein in rectal tissue. These findings demonstrate an important difference in perforin expression between CD8+ T cells in blood and mucosa. Furthermore, the relative absence of armed effector cells may serve to protect the integrity of rectal mucosa under normal conditions, but might also provide an early advantage to HIV-1 and other sexually transmitted viruses.     

Circadian rhythms of granzyme B, perforin, IFN-gamma, and NK cell cytolytic activity in the spleen: effects of chronic ethanol

Recent studies show that alterations in the body's biological rhythms can lead to serious pathologies, including cancer. Acute and chronic ethanol consumption impairs the immune system by causing specific defects in the cellular components of the innate immune response and by creating increased risk and susceptibility to infections and cancer. NK cells are critical for immune surveillance against infected and malignant cells. To assess whether NK cell function follows a circadian trend and to determine ethanol effects on this rhythm, we measured, over a 24-h period, mRNA and protein levels of granzyme B, perforin, and the cytokine IFN-gamma, as well as NK cell activity, in the splenocytes of ad libitum-fed, pair-fed, and ethanol-fed Sprague Dawley male rats. Circadian rhythms were found in mRNA and protein levels of granzyme B, perforin, and IFN-gamma. A circadian pattern was also detected in NK cell cytolytic activity. Our data further demonstrated how chronic ethanol suppressed NK cell activity by directly disrupting the circadian rhythms of granzyme B, perforin, and IFN-gamma. These findings identify the circadian functions of splenic NK cells and show the vulnerability of these rhythms to chronic ethanol.     

Circulating and liver resident CD4+CD25+ regulatory T cells actively influence the antiviral immune response and disease progression in patients with hepatitis B

CD4+CD25+ regulatory T cells (Treg) have been shown to maintain immune tolerance against self and foreign Ags, but their role in persistent viral infection has not been well-defined. In this study, we investigated whether and where CD4+CD25+ Treg contribute to the development of chronic hepatitis B (CHB). One hundred twenty-one patients were enrolled, including 16 patients with acute hepatitis B, 76 with CHB, and 29 with chronic severe hepatitis B. We demonstrated that in chronic severe hepatitis B patients, the frequencies of CD4+CD25+ Treg in both PBMC and liver-infiltrating lymphocytes were significantly increased and there was a dramatic increase of FoxP3(+)-cell and inflammatory cell infiltration in the liver compared with healthy controls. In CHB patients, circulating CD4+CD25+ Treg frequency significantly correlates with serum viral load. In acute hepatitis B patients, circulating CD4+CD25+ Treg frequency was initially low and with time, the profile reversed to exhibit an increased number of circulating Treg in the convalescent phase and restored to normal levels upon resolution. In PBMC taken from infected patients, depletion of CD4+CD25+ Treg led to an increase of IFN-gamma production by HBV-Ag-stimulated PBMC. In addition, CD4+CD25+ Treg were capable of suppressing proliferation of autologous PBMC mediated by HBV Ags, which probably reflects the generation of HBV-Ag-specific Treg in circulation and in the liver of HBV-infected patients. Together, our findings suggest that CD4+CD25+ Treg play an active role not only in modulating effectors of immune response to HBV infection, but also in influencing the disease prognosis in patients with hepatitis B.     

外周血单核细胞CD58和CD58mRNA表达与HBV感染关系的研究

为了探讨PBMCCD58(LFA3)表达与乙型肝炎病毒感染关系,应用逆转录荧光定量聚合酶链反应(RTFQPCR)法和流式细胞术分别检测43例HBV感染者(慢性肝炎轻度组12例,中度组11例,重度组10例,重型乙型肝炎组10例)和11例正常对照的外周血单核细胞(PBMC)CD58mRNA及CD58+细胞百分率的水平,同时检测血清ALT、AST水平。结果显示,乙型肝炎各组患者PBMCCD58mRNA及CD58+细胞百分率水平较正常对照组明显增高,差异均有显著性(P<0.05);CD58水平由低到高依次分别为慢性乙型肝炎轻度组、中度组、重度组及重型乙型肝炎组,各组患者间相比较,差异亦有显著性(P<0.05);乙型肝炎患者PBMCCD58mRNA及CD58+细胞百分率水平与血清ALT、AST呈显著正相关。因此,乙型肝炎患者PBMCCD58mRNA及CD58+细胞百分率水平与疾病严重程度及肝细胞损伤严重程度有关。     

Intrahepatic lymphocyte expression of dipeptidyl peptidase I-processed granzyme B and perforin induces hepatocyte expression of serine proteinase inhibitor 6 (Serpinb9/SPI-6)

Human proteinase inhibitor 9 (PI-9/serpinB9) and the murine ortholog, serine proteinase inhibitor 6 (SPI-6/serpinb9) are members of a family of intracellular serine proteinase inhibitors (serpins). PI-9 and SPI-6 expression in immune-privileged cells, APCs, and CTLs protects these cells against the actions of granzyme B, and when expressed in tumor cells or virally infected hepatocytes, confers resistance to killing by CTL and NK cells. The present studies were designed to assess the existence of any correlation between granzyme B activity in intrahepatic lymphocytes and induction of hepatic SPI-6 expression. To this end, SPI-6, PI-9, and serpinB9 homolog expression was examined in response to IFN-alpha treatment and during in vivo adenoviral infection of the liver. SPI-6 mRNA expression increased 10- to 100-fold in the liver after IFN-alpha stimulation and during the course of viral infection, whereas no significant up-regulation of SPI-8 and <5-fold increases in other PI-9/serpinB9 homolog mRNAs was observed. Increased SPI-6 gene expression during viral infection correlated with influxes of NK cells and CTL. Moreover, IFN-alpha-induced up-regulation of hepatocyte SPI-6 mRNA expression was not observed in NK cell-depleted mice. Additional experiments using genetically altered mice either deficient in perforin or unable to process or express granzyme B indicated that SPI-6 is selectively up-regulated in hepatocytes in response to infiltration of the liver by NK cells that express perforin and enzymatically active granzyme B.     

干扰素对慢性乙肝患者CD25表达及PBMC内HBV-DNA的作用

目的 探讨干扰素对慢性乙肝患者CD25的诱导及其对PBMC内的HBV-DNA的抗病毒作用.方法 将94例慢性乙肝患者分为A(48例)、B(46例)两组,分别采用IFN-α2b和常规治疗2个疗程.应用生物素-链霉亲和素(biotin-streptavidin,BSA)法检测治疗前后的及PHA体外诱导后的患者PBMC的CD...     

Virus-specific CD8+ lymphocytes share the same effector-memory phenotype but exhibit functional differences in acute hepatitis B and C

Hepatitis B and hepatitis C viruses (HBV and HCV) are both noncytopathic and can cause acute and chronic infections of the liver. Although they share tropism for the same organ, development of chronic hepatitis is much more frequent following HCV infection, suggesting different mechanisms of viral persistence. In this study, we show that circulating HBV- and HCV-specific tetramer-positive CD8 cells during the acute phase of hepatitis B and C belong almost entirely to an effector-memory subset (CCR7(-) CD45RA(-)). Despite this phenotypic similarity, HBV- and HCV-specific CD8 cells show striking functional differences. HBV-specific tetramer-positive CD8 cells express high perforin content ex vivo, expand vigorously, and display efficient cytotoxic activity and gamma interferon (IFN-gamma) production upon peptide stimulation. A comparable degree of functional efficiency is maintained after the resolution of hepatitis B. In contrast, HCV-specific CD8 cells in the acute phase of hepatitis C express significantly lower levels of perforin molecules ex vivo and show depressed CD8 function in terms of proliferation, lytic activity, and IFN-gamma production, irrespective of the final outcome of the disease. This defect is transient, because HCV-specific CD8 cells can progressively improve their function in patients with self-limited hepatitis C, while the CD8 function remains persistently depressed in subjects with a chronic evolution.     

Detection of HCV and GBV-CHGV RNA in peripheral blood mononuclear cells of patients with chronic type C hepatitis

Hepatitis C virus (HCV) and hepatitis G virus (HGV) viraemia were investigated by RT-PCR protocols in peripheral blood mononuclear cells (PBMC) of 22 patients with chronic type C hepatitis. Samplings were at basal and 4-8 months after a 12 month period of treatment with interferon-alpha. A plus strand of HCV in PBMC was detected in 8 of 21 patients (38%) (p <0.05; chi2 test) with a lack of response to therapy; a minus strand was detected in 10% of chronic type C hepatitis and 25% of the patients harboured HCV RNA in PBMC. The association with a response was nearly significant (p <0.1; chi2 test). GBV-C/HGV RNA was detected in the serum of 9 of 21 (43%) patients and in PBMC of 20% of the patients viraemic. Genomic sequences of GBVC/HGV in PBMC were found, but further investigation is needed to assess the findings reported for HCV.     

Immunogold labeling of perforin and serine esterases in granulated metrial gland cells

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are cytolytic lymphocytes known to produce a pore-forming protein, named perforin or cytolysin, that lyses target cells by creating large pores on the target plasma membrane. Besides perforin, the granules of CTL and NK cells contain a family of serine esterases. Perforin has also been localized in granulated metrial gland (GMG) cells of the murine embryo implantation site by light microscopic immunostaining. Ultrastructural immunogold labeling with antibodies against perforin and a serine esterase (MTSP 1 or granzyme A) shows that GMG cells contain both perforin and serine esterases in the fine granular matrix of their granules. Perforin has been located in all of the granules, whereas gold particles corresponding to serine esterases have been found in most of the granules. Results from the double immunogold technique indicate that perforin and serine esterases colocalize to most of the same granules in GMG cells. This study supports the view that GMG cells are related to cytolytic lymphocytes.     

Increased expression of perforin and granzyme B genes in patients with metastatic melanoma treated with recombinant interleukin-2

The frequency of peripheral blood cells expressing the perforin gene or the granzyme B gene was evaluated by in situ hybridization in nine patients suffering from metastatic melanoma and treated with recombinant interleukin-2 (rIL-2). A spontaneous expression of both genes was detected in five to seven patients, rIL-2 administration increased the frequency of positive cells in all patients (P<0.03 for each gene), the highest frequency being reached in the patients who already expressed these genes prior to rIL-2 treatment (P<0.02). Expressions of the granzyme B gene and of the perforin gene were strongly correlated before IL-2 treatment and they were similarly affected by rIL-2 administration. In contrast, their modification under treatment did not correlate with that of CD56⁺ cell counts, of natural killer activity and of sCD8 release. This indicates that perforin and granzyme B gene expressions are markers of cytotoxic cell activation independent of those previously described, and that they should be further evaluated in patients with malignancies to delineate their potential value in predicting clinical outcome.     

Genetic Diversity and Tissue Compartmentalization of the Hepatitis C Virus Genome in Blood Mononuclear Cells, Liver, and Serum from Chronic Hepatitis C Patients

The degree of genetic variability in the hypervariable region 1 of hepatitis C virus (HCV) was analyzed by cloning and sequencing HCV genomes obtained in paired samples of serum, liver tissue, and peripheral blood mononuclear cells (PBMC) from four chronic hepatitis C patients. Genetic variability in serum was higher than in liver tissue or PBMC at the level of complexity (the number of different sequences obtained from each type of tissue) as well as at the level of genetic distance between all pairs of sequences within each tissue (compared by the Student t test; P < 0.001 for two patients and P < 0.01 for another). The spectrum of viral genomes differed among the three types of tissue, as shown by segregation of sequences according to their tissue of origin in phylogenetic analysis and by statistical analysis of mean genetic distances observed between sequences obtained from different tissues (P < 0.001), but sequences from liver tissue and PBMC were more closely related to each other than to those from serum.     

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. Received: 5 December 1996 / Accepted: 16 January 1997     

Detection of diverse hepatitis C virus (HCV)-specific cytotoxic T lymphocytes in peripheral blood of infected persons by screening for responses to all translated proteins of HCV

Broadly directed hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) have been identified from liver-infiltrating lymphocytes but have been more difficult to assess in peripheral blood of infected persons. To enhance the detection of CTL from peripheral blood mononuclear cells (PBMC), we cocultured PBMC with autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines that had been infected with recombinant vaccinia virus constructs so that they expressed the entire translated polyprotein of HCV-H, a type 1a strain. These stimulated cells from HCV-infected as well as exposed seronegative persons were then cloned at limiting dilution and tested for HCV-specific CTL activity using a standard (51)Cr release assay. HCV-specific CTL were detected in PBMC from seven of nine persons with chronic hepatitis, including five of seven in whom CTL had previously been detected from liver biopsy specimens but not PBMC. In a single person with chronic HCV infection, CTL directed against as many as five different epitopes were detected in peripheral blood and were similar in specificity to those detected in liver tissue. This technique was used to evaluate eight subjects identified to be at high risk for HCV exposure due to continued injection drug abuse; no evidence of CTL in PBMC was found. We conclude that CTL can be detected in PBMC from the majority of persons with chronic HCV infection but are present at lower levels or absent in exposed but persistently seronegative persons. The high degree of concordance of HCV epitopes identified from liver and PBMC suggests that this strategy is a reasonable alternative to liver biopsy for characterizing the CTL response to HCV in chronically infected persons.     

草鱼穿孔素C端溶细胞活性分析

穿孔素的溶细胞作用是机体杀伤病毒和其他微生物感染细胞以及肿瘤细胞的一种效应机制。穿孔素在鱼类非特异性免疫中起重要作用。为了解鱼类穿孔素的功能,根据穿孔素基因序列特征,在已构建的草鱼肝肾cDNA文库中克隆了草鱼穿孔素基因C端包含1个完整蛋白激酶C保守结构域(C2)的cDNA。将该cDNA与表达载体pET32a连接并转化表达菌DE3,诱导表达。His-Bind亲和柱纯化获得了草鱼穿孔素C端表达多肽(PFP-C)。将PFP-C与兔红细胞共育,结果表明：PFP-C具有溶血功能,且在pH 7.5时活性最大,其溶血活性对Ca2+有明显的依赖性。     

Involvement of granule proteins in T-cell-mediated cytolysis

Cytolytic T lymphocytes (CTL) and large granular lymphocytes contain dense cytoplasmic granules which, when isolated, are lytic for a variety of target cells. Granule proteins are released from the effector cell upon target cell interaction, further suggesting that they play a role in the cytolytic mechanism. Major proteins in CTL granules are a family of serine esterases (granzymes) and a pore-forming protein called perforin (cytolysin). Despite structural similarities between functionally conserved regions of perforin and the ninth component of complement (C9), these two lytic molecules are clearly distinct in their mode of target cell recognition. Perforin, unlike C9, is not dependent on a protein receptor molecule but binds to the target cell membrane via phosphorylcholine in a Ca2(+)-dependent manner. Here, we discuss the stimulus-secretion model for T-cell-mediated cytotoxicity with respect to our current understanding of perforin and the granzyme proteases.     

Modulation of perforin, granzyme A, and granzyme B in murine natural killer (NK), IL2 stimulated NK, and lymphokine-activated killer cells by alcohol consumption.

Alcohol consumption in mice suppresses the cytolytic activity of natural killer (NK) and lymphokine-activated killer (LAK) cells through unknown mechanisms. Herein, we found that alcohol consumption decreased target cell-induced release of granzyme A activity in freshly isolated splenic NK cells, in NK cells stimulated for 18 h with 1000 IU/ml of interleukin 2, and in LAK cells. The total activity and protein expression of granzymes A and B also were lower in these cells than in cells isolated from water-drinking mice. Interleukin 2 increased granzyme A protein expression independent of alcohol consumption; however, this increase was associated with decreased enzyme activity. In contrast, granzyme B protein expression and enzymatic activity increased in response to interleukin 2. Perforin activity and protein expression were reduced in LAK cells generated from alcohol-consuming mice. We conclude that the mechanism underlying the suppression of NK and LAK cytolytic activity by alcohol consumption involves the collective reduction of target-induced release, activity, and expression of perforin and granular proteases.     

Perforin mediated apoptosis of cerebral microvascular endothelial cells during experimental cerebral malaria

Cerebral malaria is a serious complication of Plasmodium falciparum infection. We have investigated the role of perforin in the pathogenesis of cerebral malaria in a murine model (Plasmodium berghei ANKA (PbA) infection). C57BL/6 mice demonstrated the typical neuropathological symptoms of experimental cerebral malaria infection from day 5p.i. and became moribund on day 6p.i. This pathology was not seen in PbA-infected, perforin-deficient (pfp-/-) mice. From days 5-6p.i. onwards there was a significant increase in mRNA for granzyme B and CD8, but not CD4, in brain tissue from PbA-infected C57BL/6 and pfp-/- mouse brains. Perforin mRNA was strongly increased in the brains of PbA-infected C57BL/6 mice on day 6p.i. Immunohistochemistry revealed increased perforin staining and elevated numbers of CD8(+) cells within the cerebral microvessels in PbA-infected C57BL/6 at days 5 and 6p.i. compared with uninfected animals. At day 6p.i., there were TUNEL-positive cells and activated caspase-3 positive cells of endothelial morphology in the CNS of PbA-infected C57BL/6 mice. The TUNEL-positive cells were greatly reduced in pfp-/- mice. These results suggest that CD8(+)T lymphocytes induce apoptosis of endothelial cells via a perforin-dependent process, contributing to the fatal pathogenic process in murine cerebral malaria.