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R C Bleackley

Publications and source records attributed to R C Bleackley.

At least 19 recordsLinked to original sources
PubMed1996

An interleukin-1beta converting enzyme-like protease is a key component of Fas-mediated apoptosis.

Cytotoxic T lymphocytes (CTLs) are able to kill target cells bearing foreign antigen through two distinct mechanisms: granule- and Fas-mediated cytotoxicity. The exact events involved in the induction of target cell apoptosis remain elusive, but research indicates a role for members of the interleukin-1beta converting enzyme (ICE)/Ced-3 family of cysteine proteases. The exact nature of the protease(s) involved is yet to be determined. Here we use activity assays and peptide inhibitors of ICE/Ced-3 proteases to study their role in Fas-mediated killing. We find that while certain inhibitors block DNA fragmentation and chromium release, others do not. Most notably, potent inhibitors of CPP32 and ICE could not inhibit DNA fragmentation during all cases of Fas-mediated cytotoxicity although an "ICE" inhibitor could suppress 51Cr release. Additionally, we find that CPP32 is not cleaved in all target cells during Fas killing. Although ICE activity (as measured by a fluorogenic substrate) is present in cell lysates from anti-Fas-treated cells, we found no pro-IL-1beta-cleaving activity in these lysates. Taken together, our results suggest that an alternate pathway to DNA fragmentation exists, which does not involve CPP32 activity, and that CPP32 and ICE activities are not essential to Fas-mediated killing.

Animals↗
PubMed1996

Cleavage of CPP32 by granzyme B represents a critical role for granzyme B in the induction of target cell DNA fragmentation.

Cytotoxic T lymphocytes (CTLs) are able to recognize and destroy target cells bearing foreign antigen using one of two distinct mechanisms: granule- or Fas-mediated cytotoxicity. The exact mechanisms involved in the induction of apoptotic cell death remain elusive; however, it seems likely that a family of cysteine proteases related to interleukin-1beta converting enzyme are involved. One family member, CPP32, has been identified as an intracellular substrate for granzyme B, a CTL-specific serine protease responsible for the early induction of target cell DNA fragmentation. Here we use cytolytic cells from granzyme B-deficient mice to confirm that cleavage and activation of CPP32 represents a nonredundant role for granzyme B and that this activation plays a role in the induction of DNA fragmentation in target cells, a signature event for apoptotic cell death. A peptide inhibitor of CPP32-like proteases confirmed the function of these enzymes in fragmentation. 51Cr release was not suppressed under these conditions, suggesting that granzyme B cleavage of CPP32 is primarily involved in the induction of DNA fragmentation and not membrane damage during CTL-induced apoptosis.

Animals↗
PubMed1996

Differential inhibition of the Fas- and granule-mediated cytolysis pathways by the orthopoxvirus cytokine response modifier A/SPI-2 and SPI-1 protein.

Cytotoxic T lymphocytes are important effectors of antiviral immunity, and they induce target cell death either by secretion of cytoplasmic granules containing perforin and granzymes or by signaling through the Fas cell surface antigen. Although it is not known whether the granule-mediated and Fas-mediated cytolytic mechanisms share common components, proteinase activity has been implicated as an important feature of both pathways. The orthopoxviruses cowpox virus and rabbitpox virus each encode three members of the serpin family of proteinase inhibitors, designated SPI-1, SPI-2, and SPI-3. Of these, SPI-2 (also referred to as cytokine response modifier A in cowpox virus) has been shown to inhibit the proteolytic activity of both members of the interleukin 1 beta converting enzyme family and granzyme B. We report here that cells infected with cowpox or rabbitpox viruses exhibit resistance to cytolysis by either cytolytic mechanism. Whereas mutation of the cytokine response modifier A/SPI-2 gene was necessary to relieve inhibition of Fasmediated cytolysis, in some cell types mutation of SPI-1, in addition to cytokine response modifier A/SPI-2, was necessary to completely abrogate inhibition. In contrast, viral inhibition of granule-mediated killing was unaffected by mutation of cytokine response modifier A/SPI-2 alone, and it was relieved only when both the cytokine response modifier A/SPI-2 and SPI-1 genes were inactivated. These results suggest that an interleukin 1 beta converting enzyme-like enzymatic activity is involved in both killing mechanisms and indicate that two viral proteins, SPI-1 and cytokine response modifier A/SPI-2, are necessary to inhibit both cytolysis pathways.

Animals↗
PubMed1996

In vivo regulation of murine granzyme B gene transcription in activated primary T cells.

A murine granzyme B promoter fragment that extends 243 base pairs upstream of the transcription start site confers high levels of luciferase reporter gene activity in transient transfection assays into T cells and mouse L cell fibroblasts. This promoter fragment contains canonical binding sites for the transcription factors AP-1, core binding factor (CBF), Ikaros, and the cyclic AMP responsive element binding protein (CREB). Oligonucleotides containing the granzyme B AP-1 or CBF elements form specific complexes with proteins present in nuclear extracts from activated CD8(+) splenocytes, MTL cells, EL4 T cells, and L cells. A strong DNase1 hypersensitive site that coincides with the closely associated AP-1, CBF, Ikaros, and CRE elements is present in activated CD8(+) T cells but not in resting T cells or L cells. Both in vitro and in vivo footprints are observed at these sequence elements in activated cytotoxic T cells (CTL) but not in resting T cells. The endogenous granzyme B gene is CTL-specific as no mRNA is detectable in EL4 or L cells. We propose that a condensed chromatin structure at the granzyme B promoter is responsible for transcription factor inaccessibility and repression of transcription in non-T cells.

Animals↗
PubMed1996

Processing and activation of CMH-1 by granzyme B.

Granzyme B plays an essential role in cytotoxic T lymphocyte (CTL)-mediated cell killing. Recent studies suggest that granzyme B may exert its effect by cleaving and activating CPP32, a member of the interleukin-1 beta-converting enzyme/Ced-3 family of cysteine proteases. We have examined the processing and activation of CMH-1, a close homologue of CPP32, by granzyme B in vitro. We have found that granzyme B specifically cleaves CMH-1 at Asp198-Ser199 between the p20 and p12 and activates the cysteine protease. Cleavage between p20 and the prosequence of CMH-1 at Asp23-Ala24 is autocatalytic and is not required for CMH-1 activity in vitro. The cleavage and activation of CMH-1 by granzyme B in vitro sugge st that, in addition to CPP32, CMH-1 may also play a role in CTL-mediated cell killing.

Amino Acid Sequence↗
PubMed1996

Binding of granzyme B in the nucleus of target cells. Recognition of an 80-kilodalton protein.

Granzyme B (cytotoxic cell proteinase 1) is a serine proteinase that has been implicated in cytotoxic T lymphocyte-induced apoptosis. In order to understand how granzyme B is involved in mechanisms of target cell destruction, characterization and identification of substrates are required. We have developed an in situ binding assay using permeabilized cells and recombinant granzyme B that allows us to visualize potential substrates after immunostaining with anti-granzyme B antiserum. Confocal laser scanning microscopy and immunoelectron microscopic analyses demonstrate that granzyme B recognizes a nuclear substrate. The labeling pattern observed corresponds with regions of positive staining with uranyl acetate which binds to heterochromatin in the nucleus. Positive labeling of target cells with granzyme B is dependent on the presence of a catalytically active proteinase, since an inactive proenzyme form of granzyme B fails to give rise to any binding in the target cells. Far-Western blotting and immunoprecipitation of subcellular fractions of target cells have shown that the putative substrate of catalytically active granzyme B is an 80-kDa nuclear protein. Minor cytosolic bands of 50 and 94 kDa are also observed. A cytoplasmic band of 69 kDa is detected by both active and zymogen forms of granzyme B.

Animals↗
PubMed1996

The cell-mediated cytotoxic response to influenza vaccination using an assay for granzyme B activity.

The measurement of cytotoxic T lymphocyte (CTL) activity through 51Cr assays is a very labour intensive method for studying cytotoxicity in human CTL due to the necessary preparation of autologous targets for the assay. An assay for granzyme B, one of a family of serine proteinases implicated in the 'lethal hit' that leads to target cell lysis, is an alternative simple measure of CTL activation. We measured granzyme B activity using its both preferred and unique substrate tert-butyloxycarbonyl-Ala-Ala-Asp-thiobenzyl ester (BAADT) in peripheral blood mononuclear cells (PBMC) obtained from influenza vaccinated subjects, and stimulated with live virus. We found that granzyme B activity increases in parallel and correlates with cytolytic activity as measured by 51Cr release assays in these virus-stimulated PBMC cultures. The assay was then used to measure the cell-mediated cytotoxic response to influenza vaccination in ten healthy elderly subjects. Peak granzyme B activity (day 6) was measured in lysates of PBMC stimulated with influenza virus, obtained from study participants before and after vaccination. We found a significant increase in granzyme B activity from pre-vaccination levels to 4 weeks post vaccination (pre=2.77 U/mg protein, post=7.23 U/mg protein, p=0.002) and a subsequent decline in the activity measured at 12 weeks post vaccination (4.34 U/mg protein, p=0.0007). Due to its substrate specificity which is unique within the family of serine proteases, this assay is highly specific for granzyme B. The assay also avoids the potential hazard of radioactivity (51Cr) in the clinical laboratory and the need for a gamma counter. The assay of granzyme B activity, therefore, provides a simple, specific and responsive method for measuring changes in cell-mediated cytotoxic activity resulting from influenza vaccination.

Adult↗
PubMed1996

A physical interaction between the cell death protein Fas and the tyrosine kinase p59fynT.

The Fas antigen (Apo1/CD95) is a transmembrane protein belonging to the nerve growth factor receptor family. It is expressed on a variety of cells, including activated T lymphocytes. Ligation of Fas with its natural ligand or with anti-Fas antibodies often results in the apoptotic death of the cell, making Fas an important mediator of down-regulating immune responses. The signal transduction pathways utilized by Fas are currently unknown, although tyrosine kinase activity has recently been strongly implicated. Here, we report that the tyrosine kinase p59fyn physically associates with Fas in Fas-sensitive cells. In addition, we show that activated T lymphocytes from fyn knockout mice exhibit elevated lifespans and reduced apoptosis in vitro compared to their normal counterparts. Furthermore, activated T lymphocytes from the fyn-deficient mice are less sensitive to killing by both anti-Fas antibody and Fas-ligand cytotoxic T cells. These results suggest that p59fyn plays an important role in Fas signal transduction.

Amino Acid Sequence↗
PubMed1996

Helper and cytotoxic T lymphocyte responses to influenza vaccination in healthy compared to diabetic elderly.

This study was designed to determine the effect of Type II diabetes mellitus in older adults on two measures of the cell-mediated immune response to influenza vaccination. Twenty-two elderly persons with diabetes mellitus were compared to 20 healthy seniors, all of whom were living independently in the community. Peripheral blood mononuclear cells (PBMC) were challenged in vitro with live influenza virus, pre-vaccination and 4 and 12 weeks post-vaccination. PBMC culture supernatants were assayed for IL-2 activity as a measure of the helper T-cell response to vaccination. The cytotoxic T-lymphocyte response was determined using an assay of granzyme B activity in PBMC lysates. Initial analysis of the data showed increased IL-2 production in post-vaccination PBMC cultures from the diabetic group compared to the healthy group. However, when vaccination histories were used in an analysis of variance, it was found that the difference between the two groups was related to vaccination history. Study subjects vaccinated one year prior to participation in this study compared to those who had not been previously vaccinated, demonstrated a significantly suppressed IL-2 response to vaccination. Type II diabetes mellitus had no effect on the IL-2 response to vaccination. The granzyme B response to vaccination was not significantly affected by previous vaccination and results were similar for the healthy and diabetic elderly groups.

Aged↗
PubMed1996

Inducible nitric oxide synthase (iNOS) in pancreatic islets of nonobese diabetic mice: identification of iNOS- expressing cells and relationships to cytokines expressed in the islets.

Inflammatory cytokines and nitric oxide (NO) are candidate mediators of pancreatic islet beta-cell destruction in insulin-dependent diabetes mellitus. In this study, we used a semiquantitative PCR assay to measure levels of messenger RNA (mRNA) expression of the inflammatory cytokines, interleukin-1 alpha (IL-1 alpha), tumor necrosis factor-alpha, and interferon-gamma (IFN gamma), and of the inducible form of NO synthase (iNOS) in mononuclear leukocytes isolated from pancreatic islets of autoimmune diabetes-prone nonobese diabetic (NOD) female mice. We found that mRNA levels of iNOS, IL-1 alpha, and IFN gamma in islet mononuclear leukocytes increased from 5 weeks of age to onset of diabetes ( > 13 weeks of age). To determine whether increased iNOS, IL-1 alpha, and IFN gamma mRNA expressions were related to diabetes development, we compared mRNA levels of these molecules in mononuclear leukocytes from islets of 12 week-old diabetes-prone NOD female mice and three groups of 12-week-old mice with low diabetes risk: NOD female mice injected with complete Freund's adjuvant at 4 weeks of age, NOD male mice, and BALB/c female mice that do not develop diabetes. We found that iNOS, IL-1 alpha, and IFN gamma mRNA levels were higher in mononuclear leukocytes from islets of diabetes-prone NOD female mice than in those from mice correlated with IL-1 alpha and IFN gamma mRNA levels. By using specific antibodies and immunohistochemical methods, we localized iNOS in macrophages as well as in beta-cells of islets from diabetes-prone NOD female mice. These findings suggest that IL-1 alpha and IFN gamma may promote islet beta-cell destruction at least in part by up-regulating iNOS expression an No production by both macrophages and beta-cells in the islets of autoimmune diabetes-prone NOD mice.

Animals↗
PubMed1995

Activation of the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B.

Cytotoxic T lymphocyte (CTL)-mediated cytotoxicity represents the body's major defence against virus-infected and tumorigenic cells, and contributes to transplant rejection and autoimmune disease. During killing, CTL granules are exocytosed, releasing their contents into the intercellular space between the target cell and the effector. Perforin facilitates the entry of cytotoxic cell serine proteases, the granzymes, into the target cell, where they induce apoptotic death by an unknown pathway. Granzyme B is essential for the induction of DNA fragmentation and apoptosis in target cells, yet its substrate is unknown. Identification of the intracellular substrate for granzyme B is therefore the key to understanding the mechanism of CTL-mediated killing. Here we show that granzyme B cleaves and activates CPP32, the precursor of the protease responsible for cleavage of poly(ADP-ribose) polymerase.

Apoptosis↗
PubMed1995

Combined therapy with interleukin-4 and interleukin-10 inhibits autoimmune diabetes recurrence in syngeneic islet-transplanted nonobese diabetic mice. Analysis of cytokine mRNA expression in the graft.

Syngeneic pancreatic islet grafts in nonobese diabetic (NOD) mice elicit a cell-mediated autoimmune response that destroys the insulin-producing beta cells in the islet graft. IL-4 and IL-10 are cytokines that inhibit cell-mediated immunity. In this study, we evaluated the effects of IL-4 and IL-10 on the survival of syngeneic pancreatic islets transplanted into diabetic NOD mice. Islet grafts survived beyond 18 days and normoglycemia was maintained in 67% (10 of 15) of mice treated with IL-4 plus IL-10, but in none (0 of 20) of vehicle-injected (control) mice. Also, 40% (6 of 15) of the mice treated with IL-4 plus IL-10 were normoglycemic at 30 days after transplantation, compared with 14% (1 of 7) of the mice treated with IL-4 alone, 8% (1 of 13) of the mice treated with IL-10 alone, and none (0 of 20) of the control mice. Histological examination of grafts at 10 days after transplantation revealed peri-islet accumulations of mononuclear leukocytes and intact islet beta cells in grafts from IL-4 plus IL-10-treated mice, whereas islets were infiltrated by leukocytes and the beta cell mass was greatly reduced in grafts from control mice. Polymerase chain reaction (PCR) analysis of cytokine mRNA expression in the grafts revealed higher levels of IL-2, IFN gamma, and IL-10 mRNA in grafts of diabetic compared with normoglycemic control mice, whereas IFN gamma and TNF alpha mRNA levels were significantly decreased in grafts of IL-4 plus IL-10-treated mice compared with either normoglycemic or diabetic control mice. These results suggest that T helper (Th)1 cells and their cytokine products (IL-2, IFN gamma, and TNF alpha) may promote islet beta cell destructive insulitis and autoimmune diabetes recurrence in syngeneic islet-transplanted NOD mice, and that administration of IL-4 plus IL-10 may inhibit diabetes recurrence by suppressing Th1 cytokine production in the islet grafts.

Animals↗
PubMed1995

Granzyme B is inhibited by the cowpox virus serpin cytokine response modifier A.

The ability of cytolytic cells to cause apoptosis in target cells is in part due to the action of the serine proteinase granzyme B. We demonstrate that granzyme B is inhibited, with an association rate constant of 2.9 x 10(5) M-1 s-1, by the cowpox viral serpin cytokine response modifier A (CrmA). Previously we have shown CrmA to be an inhibitor of the cysteine proteinase interleukin-1 beta-converting enzyme (ICE). Thus the mechanism of CrmA involves the unusual ability to efficiently inhibit proteinases from two distinct catalytic classes, in this case serine and cysteine proteinases. Granzyme B and ICE are both used to combat viral infection, and we propose that cowpox virus uses CrmA to evade the contribution of these two proteinases. Thus, through CrmA, the virus may influence two of the pathways normally used to kill virus-infected cells: acting on endogenous proteinases such as ICE and on exogenous proteinases delivered by cytotoxic lymphocytes to infected cells.

Binding, Competitive↗
PubMed1995

IFN-gamma gene expression in pancreatic islet-infiltrating mononuclear cells correlates with autoimmune diabetes in nonobese diabetic mice.

Insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice results from selective destruction of pancreatic islet beta-cells following islet infiltration by mononuclear leukocytes. Cytokines produced by islet-infiltrating mononuclear cells may be involved in beta-cell destruction. Therefore, we analyzed cytokine mRNA expression, by reverse-transcriptase PCR (RT-PCR) assay, in mononuclear leukocytes isolated from pancreatic islets of four groups of mice: diabetes-prone female NOD mice; female NOD mice protected from diabetes by injection of CFA at an early age; male NOD mice with a low diabetes incidence; and female BALB/c mice that do not develop diabetes. We found that mRNA levels of IL-1 beta, IL-2, IL-4, IL-10, and IFN-gamma in mononuclear cells from islets of diabetes-prone female NOD mice increased progressively as these cells infiltrated the islets from age 5 wk to diabetes onset (> 13 wk). However, only IFN-gamma mRNA levels were significantly higher in islet mononuclear cells from 12-wk-old diabetes-prone female NOD mice than from less diabetes-prone NOD mice (CFA-treated females, and males) and normal mice (BALB/c). In contrast, IL-4 mRNA levels were lower in islet mononuclear cells from diabetes-prone female NOD mice than from NOD mice with low diabetes incidence (CFA-treated females and males). Splenic cell mRNA levels of IFN-gamma and IL-4 were not different in the four groups of mice. These results suggest that islet beta-cell destruction and diabetes in female NOD mice are dependent upon intra-islet IFN-gamma production by mononuclear cells, and that CFA-treated female NOD mice and male NOD mice may be protected from diabetes development by down-regulation of IFN-gamma production in the islets.

Aging↗
PubMed1995

Proteinases are involved in both DNA fragmentation and membrane damage during CTL-mediated target cell killing.

A number of inhibitors with different specificities were used to probe the involvement of proteinases in the mechanism of cytotoxic T lymphocyte (CTL)-mediated lysis. N-Acetyl-L-tyrosine ethyl ester (ATEE) and N-benzoyl-L-arginine ethyl ester (BAEE) are reversible substrate inhibitors of proteinases with trypsin-like and chymotrypsin-like specificities, respectively. BAEE did not prevent either the chromium release or DNA fragmentation induced in mouse tumor target cells by a mixed lymphocyte population. In contrast, ATEE inhibited both processes. The irreversible proteinase inhibitor 3,4-dichloroisocoumarin (DCI) also blocked both chromium release and DNA fragmentation, but at significantly lower concentrations than ATEE. More importantly, chromium release was more susceptible to inhibition by DCI than DNA fragmentation. Addition of a combination of the endonuclease inhibitor aurintricarboxylic acid plus DCI resulted in virtually complete inhibition of both DNA fragmentation and chromium release when the drugs were added at the beginning of the incubation period. In contrast addition of DCI 15 or 30 min following initiation of the lytic cycle abolished the affect of DCI on fragmentation, but not lysis. A model which suggests a dual role for the proteinases in CTL-mediated target cell death is presented. First, proteinases are involved in the initiation of DNA fragmentation. Second, they have an ongoing function in membrane damage.

Animals↗
PubMed1995

Vaccine-related determinants of the interleukin-2 response to influenza vaccination in healthy young and elderly adults.

This study was designed as a parallel-groups comparison of trivalent preparations of split-virus vaccine (SVV) and whole-virus influenza vaccine (WVV) in healthy young and elderly adults. Interleukin-2 (IL-2) was measured in the supernatants of peripheral blood mononuclear cell (PBMC) cultures stimulated with live virus preparations of the two strains of influenza A contained in the vaccine. The duration of increased in vitro IL-2 production after vaccination was significantly different between the two strains of virus. A/Beijing/353/89 resulted in an IL-2 response that was quite short (< 7 weeks) while the response to A/Texas/16/89 was much more prolonged (> 12 weeks). In the first 12 weeks after vaccination, there was no difference in IL-2 levels between SVV and WVV recipients in either the young or elderly groups. The duration of the response to A/Texas/16/89 was slightly longer in WVV recipients as measured at 26 weeks postvaccination. The viral strain contained in the vaccine appears to be an important variable in determining the duration of the IL-2 response to vaccination.

Adult↗
PubMed1994

The cytotoxic T cell proteinase granzyme B does not activate interleukin-1 beta-converting enzyme.

Murine granzyme B (cytotoxic cell proteinase-1 (CCP1)) is a member of a family of seven serine proteases found in cytoplasmic granules of cytotoxic T lymphocytes (CTLs). Evidence has suggested that it is involved in target cell DNA fragmentation during CTL-mediated cytotoxicity, although intracellular substrates for granzyme B have not yet been identified. The substrate specificity of granzyme B, requiring an aspartic acid residue at site P1, is unique among eukaryotic serine proteases and is shared with only one other known eukaryotic protease, interleukin-1 beta-converting enzyme (ICE). ICE is responsible for processing pro-interleukin-1 beta to produce biologically active interleukin-1 beta and is itself synthesized as an inactive precursor. Recent evidence has suggested a role for ICE in programmed cell death, which led to a model for CTL-mediated cytotoxicity. In this proposal granzyme B activates ICE in the target cell by proteolytically processing the ICE precursor, resulting in active ICE heterodimer that induces apoptosis in the target cell. We have isolated the cDNA encoding murine ICE and generated in vitro translated ICE precursor. Using lysates from COS cells expressing granzyme B we show that ICE precursor is not a substrate for granzyme B and propose an alternate mechanism for CTL-mediated cytotoxicity.

Animals↗