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PubMed1992

Immunohistochemical identification of cytotoxic lymphocytes using human perforin monoclonal antibody.

Perforin is a potent cytolytic pore-forming protein expressed in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. A new monoclonal antibody raised against human perforin was used to detect both in vitro and in vivo perforin expression in cytotoxic cells. Immunohistochemical analysis of human peripheral blood mononuclear cells cultured in recombinant interleukin-2 (rIL-2) showed strong granular cytoplasmic staining of the IL-2 activated cytotoxic cells. Fresh-frozen tissue sections from patients with heart allograft rejection were also stained. Strong granular cytoplasmic staining of the mononuclear inflammatory infiltrate characteristic for perforin in cardiac allograft rejection was observed. The detection and quantitative analysis of perforin-associated cytotoxic cells by the human anti-perforin monoclonal antibody will help to evaluate the significance of these functionally distinct cytotoxic cells in human tissue.

Antibodies, Monoclonal↗
PubMedDate not supplied

Immunoexpression of perforin and granzyme B on infiltrating lymphocytes in human renal acute allograft rejection.

Graft destruction can be effected by direct cell-to-cell contact between activated effector T cell and a target graft resulting in delivery of cytotoxic molecules. Perforin and granzyme B can be used as activation markers for cytotoxic cells in allograft tissue. The aim of the study was to determine the immunoexpression of perforin and granzyme B by immune cells infiltrating renal tissue during acute allograft rejection and to evaluate any correlation between the phenotype of infiltrating lymphocytes and cells expressing cytotoxic granules as well as the severity of graft damage as defined by Banff 97 criteria. Immunoperoxidase staining was carried out using monoclonal antibodies anti-perforin, -granzyme B, -CD3 and -CD8 on renal allograft biopsy specimens from twenty one patients with acute renal transplant rejection: Banff 97 IA (n = 11) and Banff 97 IB (n = 10). As a control 11 biopsy specimens of renal transplant patient without any signs of rejection were used. All allograft biopsy specimens with acute renal transplant rejection contained a high number of CD3+ T cells (Banff IA: 437.4 +/- 154.4 and Banff IB: 825 +/- 339.9 vs 123.4 +/- 52.5 in controls) and CD8+ T lymphocytes (Banff 97 IA: 177.6 +/- 89.2 and Banff IB: 293.2 +/- 112.4 vs 64.2 +/- 37.1 in controls). Immunostaining for granzyme B and perforin was negative in controls. The immunopositivity for perforin was similar in Banff IA and Banff IB acute allograft rejection (1.5 +/- 0.6 vs 1.8 +/- 0.8, respectively). Granzyme B+ cell count was significantly higher in severe rejection group Banff IB (128.3 +/- 74.3) than in Banff IA group (48.2 +/- 18.3). Moreover, in acute allograft rejection Banff IB the number of granzyme B+ cells and perforin+ cells was correlated with the number of CD8+ T cells. In conclusion, our results suggest that in acute tubulointerstitial allograft rejection activated cytotoxic T lymphocytes play a major role. The strong immunopositivity for granzyme B on infiltrating cells in renal transplant tissue is suggested as a marker of severity of graft damage.

Adult↗
PubMed2004

[Perforin and granzyme B expression in liver allograft rejection].

OBJECTIVE: To investigate the expression of perforin and granzyme B in rejection response following liver transplantation, and evaluate their roles to be used as predictive markers of rejection. METHODS: The expression of perforin and granzyme B in liver biopsies obtained from liver allograft recipients was determined by immunohistochemistry. Biopsies were classified into two groups-no evidence of rejection and rejection-according to Histopathologic criteria. The relationship between the perforin/granzume B expression and acute rejection was analyzed. RESULTS: From 19 patients, thirty-five liver biopsies were obtained after liver transplantation. Among them, nineteen samples were diagnosed as rejection response. The frequencies of perforin and granzyme B expression in rejection group were 100% (19/19) and 94.7% (18/19), respectively. While those in no rejection group were 25.0% (4/16) and 12.5 (2/16), respectively. In most rejected samples, perforin and granzyme B were expressed simultaneously. Only three samples showed perforin expression alone, while no samples demonstrated granzyme B expression alone. There was a close relationship between perforin/granzyme B expression and liver allograft rejection. CONCLUSION: Perforin and granzyme B expression seemed to be related to the development of acute rejection following liver transplantation, and might be served as sensitive and reliable markers in diagnosing acute rejection in early stage.

Adult↗
PubMed1991

Rapid loss of perforin and serine protease RNA in cytotoxic lymphocytes exposed to sensitive targets.

We have previously reported that cytotoxic lymphocytes, when exposed to sensitive target cells, temporarily lose their lytic potential. The mechanism leading to this loss of lytic activity is still unknown but it is reversible and the lytic potency can be recovered when the effector cells are incubated with interleukin-2 (IL-2) for 12-14 hr. In this study, we have investigated the regulation of RNA coding for perforin and for two serine proteases, HSP1 and HSP2, in cytotoxic lymphocytes exposed to sensitive targets. Perforin and the two serine proteases are contained in granules of major histocompatibility complex (MHC)-restricted and non-MHC-restricted cytotoxic lymphocytes, but their exact role in the lytic mechanism is still debated. Here we used four different human cytotoxic lymphocytes (CTL) as effector cells: an MHC-restricted CTL (SG-CTL), a non-MHC-restricted CTL (IE6), a natural killer (NK)-like cell line (3.3) and lymphokine-activated killer (LAK) cells. In all effector cells we observed a rapid loss of perforin and of serine protease RNAs within 5 min following the addition of sensitive targets. The effector cells recovered the RNA messages as early as 30 min, although the kinetics of recovery was faster with CTL than with NK-like or LAK effector cells. When we exposed the effector cells to resistant targets we did not detect any loss of perforin or serine protease RNAs. Incubation of the effector cells with cycloheximide, prior to the addition of sensitive targets, did not block message loss, indicating that de novo protein synthesis was not required in this process. Cycloheximide treatment, however, inhibited the recovery of perforin and serine protease RNAs. Taken together, our results indicate that the target-mediated loss of lytic activity in cytotoxic lymphocytes may be a consequence of the down-regulation of perforin or of serine protease transcripts, or both.

Blotting, Northern↗
PubMed1991

Subcellular localization of perforin and serine esterase in lymphokine-activated killer cells and cytotoxic T cells by immunogold labeling.

CTL, NK cells, and lymphokine-activated killer (LAK) cells are cytolytic lymphocytes known to produce a pore-forming protein, named perforin or cytolysin, that lyses target cells by forming large pores on the plasma membrane of the target cell. Other proteins besides perforin are found in the cytoplasmic granules of effector lymphocytes, and these include a family of serine esterases. Ultrastructural immunogold labeling studies with antibodies against perforin and a serine esterase (MTSP-1, also known as granzyme A and SE-1) show that all the granules of LAK cells and a CTL cell line contain perforin and serine esterase. For both LAK cells and CTL, perforin has been located mostly in the fine granular matrix of the granules, whereas gold particles corresponding to serine esterase have been found in both the matrix and the cap regions of the granules. Results from double immunogold labeling indicate that perforin and serine esterase colocalize to the same granules.

Animals↗
PubMed1990

In vivo expression of perforin by natural killer cells during a viral infection. Studies on uveitis produced by herpes simplex virus type I.

A potent cytolytic pore-forming protein (PFP, perforin, or cytolysin) is associated with the cytoplasmic granules of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. The role of PFP/perforin in cytolytic reactions carried out in vivo is still unclear. Here, the authors performed immunohistochemical analysis using antibodies monospecific for perforin and made use of a murine uveitis model produced by intracameral inoculation of herpes simplex virus I (HSV-I). The main cell infiltrate found in the anterior segment of virus-inoculated eyes consisted of Thy-1+/asialo GM1+/CD8-/CD4- cells, presumably representing NK cells. Perforin staining was detected mainly in cells bearing this phenotype. Perforin was only detected in cells displaying the large granular lymphocyte morphology. A small number of perforin-positive cells (less than 5%) colabeled as CD8+, indicating that these cells could have belonged to the CTL lineage. These observations show for the first time the presence of perforin-containing NK cells in tissues of animals undergoing acute viral infections.

Animals↗
PubMed1988

Target cell lysis by cytotoxic T lymphocytes that lack detectable hemolytic perforin activity.

When mouse target cells are subjected to cytolytic attack by mouse CTL cell lines that have been cultured for many months in high levels of IL-2, and have abundant perforin-rich secretory granules, they exhibit two prominent changes: 1) rapid and massive increase (greater than 10-fold) in intracellular Ca2+ concentration and 2) fragmentation of DNA into nucleosome-sized fragments. We show here that when the same target cells are subjected to cytolytic attack by perforin-deficient CTL, either human CTL or primary mouse CTL from peritoneal exudates, the same changes are observed, suggesting that perforin-rich and perforin-deficient CTL kill their target cells by similar (if not identical) mechanisms. It is possible that perforin-deficient CTL produce enough perforin to destroy target cells but not enough to be detected by currently available methods.

Animals↗
PubMed1995

Granzyme B and perforin lytic proteins are expressed in CD34+ peripheral blood progenitor cells mobilized by chemotherapy and granulocyte colony-stimulating factor.

Granzyme B and perforin are cytoplasmic granule-associated proteins used by cytotoxic T lymphocytes and natural killer (NK) cells to kill their targets. However, granzyme B gene expression has also been detected in a non-cytotoxic hematopoietic murine multipotent stem cell line, FDCP-Mix. The objective of the present study was to investigate whether granzyme B and perforin could be expressed in human hematopoietic CD34+ cells and if present, discover what their physiologic relevance could be. The primitive CD34+ human cell line KG1a was investigated first and was found to express granzyme B and perforin. Highly purified hematopoietic stem/progenitor cells were then selected using the CD34 surface antigen as marker. Steady-state bone marrow (BM) CD34+ cells did not contain these proteins. Peripheral blood (PB) CD34+ cells, which had been induced to circulate, were also analyzed. After chemotherapy (CT) and granulocyte colony-stimulating factor (G-CSF) treatment, CD34+ cells strongly expressed mRNAs and proteins of granzyme B and perforin. In contrast, CD34+ cells mobilized by G-CSF alone were negative. Western blot analysis further showed that granzyme B and perforin proteins were identical in CD34+ cells and activated PBLs. Such proteins might be implicated in the highly efficient migration of CD34+ stem/progenitor cells from BM to PB after CT and G-CSF treatment. The cellular adhesion mechanisms involved in the BM homing of CD34+ cells are disrupted at least temporarily after CT. The Asp-ase proteolytic activity of granzyme B on extracellular matrix proteins could be used by progenitor cells for their rapid detachment from BM stromal cells and perforin might facilitate their migration across the endothelial cell barrier.

Antigens, CD34↗
PubMed1996

Perforin, Fas ligand, and tumor necrosis factor are the major cytotoxic molecules used by lymphokine-activated killer cells.

Lymphokine-activated killer (LAK) cells generated from perforin knockout mice possess significantly reduced cytotoxicity against a panel of tumor target cell lines, with some tumor cells being lysed exclusively by the perforin pathway. LAK cells are also capable of Fas ligand-mediated cytotoxicity. LAK cells generated from mice deficient in both perforin and Fas ligand (PKO/gld) were not cytolytic in short term cytotoxicity assays, demonstrating that perforin and Fas ligand are required for acute target cell lysis. However, PKO/gld LAK cells were cytotoxic in long term cytotoxicity assays against TNF-sensitive tumor lines, and this cytotoxicity was completely inhibited by neutralizing TNF Abs. This potent TNF cytotoxicity has not been fully appreciated previously because of the presence of dominant-acting perforin and Fas ligand in acute tumor cell lysis. TNF-based cytotoxicity by PKO/gld LAK was both soluble and membrane bound, and both forms of TNF were constitutively expressed. Thus, LAK cells are armed with at least three cytotoxic molecules: perforin, Fas ligand, and TNF.

Animals↗
PubMed1996

A critical role of Sp1- and Ets-related transcription factors in maintaining CTL-specific expression of the mouse perforin gene.

This study was designed to determine the potential cis-elements involved in transcriptional regulation of the mouse perforin gene. DNase I hypersensitive site (DHS) mapping revealed that the perforin locus contained six DHS within 7.0 kb of the 5' upstream sequence (-7.0 kb) and two DHS in intron 2. The six 5' upstream and one intronic DHS were detected in only perforin-expressing lymphocytes. Chloramphenicol acetyltransferase (CAT) activities directed by 5' upstream promoter were detected preferentially in perforin-expressing cell lines. A construct termed PFP5a containing -795 bp exhibited the highest CAT activity, and PFP9a20 containing only -73 bp also produced significantly high CAT activity in CTLL-R8 cells. The proximal region in PFP9a20 contained two potential Sp1 binding sites (GC box and GT box) and one Ets binding site (EBS). Electrophoretic mobility shift assay showed that each of the cis-elements bound specific protein factors. When single-point mutation was introduced to each GC box, EBS, and GT box in PFP9a20, at least 3-fold less CAT activity was observed in CTLL-R8 cells. To confirm the importance of the three cis-acting elements in the perforin gene expression, point mutation was introduced again to each proximal GC box, EBS, and GT box of PFP5a. The point mutations resulted in a 2.5- to 3-fold reduction of CAT activity. The results suggest that a combination of the three proximal cis-acting elements may constitute a minimal region responsible for CTL-specific expression of perforin.

Animals↗
PubMed1997

Non-killer cell-specific transcription factors silence the perforin promoter.

Perforin is a pore-forming killer protein exclusively expressed by cells functionally defined as cytolytic lymphocytes. Previous reporter gene investigations have indicated that the cell-type-specific expression of the perforin gene is determined by the promoter and upstream region but the respective cis- and trans-acting molecules remain to be identified. In this investigation, we differentially display DNA-protein interactions of perforin-positive vs perforin-negative cell types and functionally test their biological relevance. Our results provide molecular evidence for the transcriptional repression of the perforin gene in non-killer cells by two novel regulatory elements. One of the respective transcription factors that are exclusively expressed by non-killer cells appears to be an Ets family member. Thus, the development of cells expressing perforin, and perhaps cytolytic lymphocytes in general, may involve a shutdown of the genes for these proteins.

Animals↗
PubMed1997

Ca2+-dependent, Fas- and perforin-independent apoptotic death of allografted tumor cells by a type of activated macrophage.

Both perforin- and Fas ligand (FasL)-deficient CTLs show impaired lytic activity toward most target cells. In this study, we examined whether these molecules could be involved in the cell-to-cell contact-dependent cytotoxicity mediated by macrophages infiltrating into the rejection site of allografted Meth A fibrosarcoma cells. FasL-expressing lymph node cells from MRL-lpr/lpr mice were inactive toward Meth A tumor cells. In C3H/HeJ-gld/gld (abnormal FasL) mice, allograft-induced macrophages (AIM) were cytotoxic against Meth A cells expressing no Fas Ag. Furthermore, allografted Meth A tumor cells were acutely rejected by both C3H/HeJ-gld/gld and control C3H/HeJ mice, indicating that the cytotoxic activity of AIM against Meth A tumor cells was Fas/FasL independent. On the other hand, the cytotoxic activity of AIM against the allografts was dose dependently inhibited by EGTA; and the suppression was restored by the addition of Ca2+, but not Mg2+, implying the involvement of perforin in the cytotoxicity. In the perforin-deficient mice, however, AIM were cytotoxic against Meth A tumor cells; and allografted Meth A tumor cells were acutely rejected by both perforin-deficient and control B6 mice, indicating a perforin-independent cytotoxicity. Thus, through a yet unknown mechanism, AIM induced the apoptotic death of allografted Meth A tumor cells. These results indicate that AIM-mediated cytotoxicity against allografted Meth A tumor cells is Ca2+ dependent, and that an attack by AIM results in the apoptotic death of allografted Meth A tumor cells through a third mechanism, one other than the Fas/FasL- and perforin-based pathways.

Animals↗
PubMed1998

Perforin protects against autoimmunity in lupus-prone mice.

The roles of cytolytic regulatory mechanisms in the immune system of lupus-prone mice were examined in perforin-deficient animals bearing functional or defective (lpr) Fas Ag (CD95). Perforin-deficient Fas+ animals developed accelerated autoimmunity, characterized by increased hypergammaglobulinemia, autoantibody production, and immune deposit-related end-organ disease compared with perforin-intact counterparts. In comparison, perforin-deficient lpr animals had accelerated mortality compared with perforin-intact lpr mice, associated with the abnormal accumulation of CD3+CD4-CD8- alphabeta T cells in conjunction with unaltered hypergammaglobulinemia, autoantibody production, and immune complex renal disease. These results indicate that cytolytic lymphoid regulation plays critical roles in the immune homeostasis of lupus-prone animals, and identify perforin-mediated cytotoxicity as a specific mechanism in the regulation of systemic autoimmunity.

Animals↗
PubMed2002

Fas-ligand and perforin expression in infiltrating cytotoxic T lymphocytes in the liver of chronic hepatitis C.

Cytotoxic T cells (CTLs) are believed to play an important role in the pathogenesis of chronic hepatitis C based on histological findings of the liver and in vivo experiments. Fas-ligand-Fas and perforin dependent pathways are two major killing systems when CTLs induce their target-cell death. Thus, the present study attempts to determine whether these pathways are activated, and if they are, how they are related in chronic hepatitis C. To investigate the expression of Fas-ligand and perforin, we performed double immunofluorescent staining of liver biopsy specimens from patients with chronic hepatitis C. Fas-ligand and perforin expression was observed in mononuclear cells, and the partial coexistence of the two proteins was observed. Cells expressing both proteins were positive for CD45RO(+) T cells (active T cells), whereas cells expressing perforin were negative for CD68 (macrophages). In the cases which had sustained negative HCV-RNA over 6 months after interferon treatment, Fas-ligand was not expressed, although perforin was slightly detectable. To quantitatively assess the balance of these pathways, hepatic mRNAs of Fas-ligand and perforin were measured by quantitative RT-PCR. The ratio of Fas-ligand-mRNA/perforin-mRNA was significantly correlated with serum alanine aminotransferase (ALT) levels (r=0.913). These results suggest that both pathways are activated in chronic hepatitis C and that Fas-ligand-Fas pathway may be predominant in active inflammation.

Journal Article↗
PubMed1999

Expression of perforin and granzyme B mRNA in judgement of immunosuppressive effect in rat liver transplantation.

AIM:To explore the expression of perforin and granzyme B genes mRNAto judge the effect of immunosuppression in acute rejection of liver transplantation.METHODS: The expression of perforin and granzyme B genes mRNA was examined by reverse transcription-polymerase chain reaction (RT-PCR) in hamster to rat liver grafts under the immunosuppression of cyclosporine or/and splenectomy. Histological findings were studied comparatively.RESULTS:Cyclosporine could obviously decrease the cellular infiltration, and completely repress the expression of mRNA for perforin and granzyme B, but could not change severe hepatocyte necrosis and hemorrhage. Splenectomy could significantly lighten hepatocyte necrosis, and completely eliminate hemorrhage,but not affect the cellular infiltration and the expression of perforin and granzyme B genes mRNA. Cyclosporine or splenectomy alone could not prolong the survival time, however, their combination could completely repress the rejection of liver grafts. The survival time of animals were significantly prolonged (37.1 days). The architecture of hepatic lobules was preserved. There were slight cellular infilt-ration in the portal tracts and no expression of perforin and granzyme B genes mRNA could been seen in three weeks after transplantation.CONCLUSION:Perforin and granzyme B genes are valuable in judging the effect of immunosuppression in liver transplantation.

Journal Article↗
PubMed1999

Homeostatic regulation of CD8+ T cells by perforin.

To prevent uncontrolled expansion, the massive proliferation of T cells during an acute immune response has to be followed by controlled deletion. Here we show that similar to Fas, perforin is not only an important effector molecule of cytotoxic T lymphocytes (CTL) but also involved in down-regulating peripheral T cells. Mice deficient for both the CTL effector molecule perforin and the apoptosis-inducing Fas ligand spontaneously develop infiltration of highly activated CD8(+) T cells in kidney and liver and die between 5 and 12 weeks of age. Injection of staphylococcal enterotoxin B (SEB) into perforin-deficient mice results in dramatically increased selective expansion and prolonged persistence of CD8(+), but not CD4(+), SEB-reactive T cells. Also, secondary immunization of TCR transgenic perforin-deficient mice with the lymphocytic choriomeningitis virus glycoprotein-derived epitope peptide leads to an increased proliferation of transgenic CD8(+) T cells, that is not explained by failure to deplete professional antigen-presenting cells. These results point to a novel mechanism of T cell homeostasis in which the acquisition of perforin-dependent cytotoxic activity regulates the expansion and persistence of CD8(+) effector T cells in vivo.

Animals↗
PubMed2001

Perforin-independent regulation of dendritic cell homeostasis by CD8(+) T cells in vivo: implications for adaptive immunotherapy.

We investigated here the effects of perforin on CTL responses during interaction of dendritic cells (DC) with cytotoxic T lymphocytes in vivo. Using MHC class I tetramers complexed with the immunodominant CTL epitope of the lymphocytic choriomeningitis virus glycoprotein (LCMV-GP33), we followed the kinetics of DC-induced CTL responses. GP33-presenting DC induced rapid primary expansion of both perforin-competent and -deficient CTL with similar kinetics. Secondary CTL responses in perforin-deficient and normal control mice after DC-booster immunization were more rapid than the primary responses, but never reached the high initial levels, suggesting that reactivated memory CTL eliminated the antigen-presenting DC and thereby limited the booster effect. Whereas killingof DC in vitro was strictly dependent on perforin, elimination of GP33-presenting DC by CTL in vivo was largely independent of perforin and Fas. Taken together, these results suggest that control of DC homeostasis by CTL, i. e. elimination of DC by the effector cells they had elicited, is controlled via multiple and probably redundant signals and represents an important fail-safe mechanism to avoid exaggerated CTL responses.

Animals↗
PubMed1994

Granzyme B and perforin can be used as predictive markers of acute rejection in heart transplantation.

We have investigated perforin and granzyme B expression in graft-infiltrating lymphocytes of patients who underwent heart transplantation. Those proteins are commonly present in the cytoplasmic granules of cytotoxic T lymphocytes and are released upon effector-target cell interaction. From 28 patients 103 endomyocardial biopsies were obtained and examined by histology and immunocytochemical analysis using relevant monoclonal antibodies. We found that "high" biopsy histological grades were associated with perforin and granzyme B expression in graft-infiltrating lymphocytes of patients with acute severe rejection crisis. In contrast, these markers were not detected in patients without rejection or during graft stabilization. Interestingly, in patients with mild rejection and "low" histological grades, two groups could be distinguished with a differential expression of the two intracytoplasmic proteins. The presence of perforin and granzyme B-expressing cells was found to be predictive of rapid progression to severe rejection, so that this situation required additional treatment; in contrast, their absence seemed to correlate with a good graft outcome without additional treatment. Moreover, perforin and granzyme B expression seemed to be down-regulated by immunosuppressive drugs, which coincided with graft stabilization. In conclusion, our data suggest that detection of granzyme B and perforin in graft-infiltrating lymphocytes might be helpful for routinely monitoring heart transplant patients.

Acute Disease↗