Proteinase Inhibitor 9 Research Articles

Peptide-stimulated T cells bypass immune checkpoint inhibitor resistance and eliminate autologous microsatellite instable colorectal cancer cells

Two hypermutated colon cancer cases with patient-derived cell lines, peripheral and tumor-infiltrating T cells available were selected for detailed investigation of immunological response.T cells co-cultured with autologous tumor cells showed only low levels of pro-inflammatory cytokines and failed at tumor recognition. Similarly, treatment of co-cultures with immune checkpoint inhibitors (ICI) did not boost antitumor immune responses. Since proteinase inhibitor 9 (PI-9) was detected in tumor cells, a specific inhibitor (PI-9i) was used in addition to ICI in T cell cytotoxicity testing. However, only pre-stimulation with tumor-specific peptides (cryptic and neoantigenic) significantly increased recognition and elimination of tumor cells by T cells independently of ICI or PI-9i.We showed, that ICI resistant tumor cells can be targeted by tumor-primed T cells and also demonstrated the superiority of tumor-naïve peripheral blood T cells compared to highly exhausted tumor-infiltrating T cells. Future precision immunotherapeutic approaches should include multimodal strategies to successfully induce durable anti-tumor immune responses.

The biological function of Serpinb9 and Serpinb9-based therapy.

Recent breakthroughs in discovering novel immune signaling pathways have revolutionized different disease treatments. SERPINB9 (Sb9), also known as Proteinase Inhibitor 9 (PI-9), is a well-known endogenous inhibitor of Granzyme B (GzmB). GzmB is a potent cytotoxic molecule secreted by cytotoxic T lymphocytes and natural killer cells, which plays a crucial role in inducing apoptosis in target cells during immune responses. Sb9 acts as a protective mechanism against the potentially harmful effects of GzmB within the cells of the immune system itself. On the other hand, overexpression of Sb9 is an important mechanism of immune evasion in diseases like cancers and viral infections. The intricate functions of Sb9 in different cell types represent a fine-tuned regulatory mechanism for preventing immunopathology, protection against autoimmune diseases, and the regulation of cell death, all of which are essential for maintaining health and responding effectively to disease challenges. Dysregulation of the Sb9 will disrupt human normal physiological condition, potentially leading to a range of diseases, including cancers, inflammatory conditions, viral infections or other pathological disorders. Deepening our understanding of the role of Sb9 will aid in the discovery of innovative and effective treatments for various medical conditions. Therefore, the objective of this review is to consolidate current knowledge regarding the biological role of Sb9. It aims to offer insights into its discovery, structure, functions, distribution, its association with various diseases, and the potential of nanoparticle-based therapies targeting Sb9.

Peripheral leucocytes and tissue gene expression of granzyme B/perforin system and serpinB9: Impact on inflammation and insulin resistance in coronary atherosclerosis.

The imbalance between proapoptotic granzyme B (GZB)/perforin (PRF) system and proteinase inhibitor-9 (PI-9; serpinB9); the only known inhibitor of human GZB, has been demonstrated in atherosclerosis. However, their role in atherosclerosis with the impact of type 2 diabetes mellitus (DM) as well as their contribution to hallmarks of atherosclerosis is not clear. ELISA for serum insulin, high sensitivity C-reactive protein (hsCRP) and GZB levels in atherosclerotic coronary artery diseases (CAD) patients were estimated in comparison to apparently healthy controls, while GZB, PRF and PI-9 mRNA expression levels were quantified by Taqman RT-PCR in both peripheral leucocytes and atherosclerotic tissues. Atherosclerotic patients showed significantly higher insulin, hsCRP and GZB levels than controls. There was a significant increase in GZB mRNA expression and significant reduction in PI-9 mRNA in both patient peripheral leucocytes and atherosclerotic lesions, while PRF mRNA increased significantly only in atherosclerotic tissues. PI-9 mRNA levels were significantly lower in patients with diabetes than patients without diabetes. In contrast to positive modulating effect of GZB, regression analysis revealed negative modulating effect of PI-9 on inflammation and insulin resistance. Circulating PI-9 mRNA was inversely contributed to CAD severity. GZB and PI-9 could be effective modulators for inflammation and insulin resistance in atherosclerosis.

Presence of proteinase inhibitor‐9 and granzyme B in healthy and pathological human corneas

SummaryAim: To determine whether proteinase inhibitor‐9 (PI‐9) and granzyme B (GrB) are expressed in normal and pathological corneas.Methods: Cryosections and endothelial, epithelial and conjunctival imprints of 12 human cadaverous corneo‐scleral discs and 13 pathological corneas (corneal melting and herpes virus keratitis) were used. Protein localization was examined using fluorescent and enzymatic immunohistochemistry, and mRNA expression was determined by qRT‐PCR.Results: PI‐9 is predominantly expressed in the superficial and suprabasal epithelial layers of the cornea and conjunctiva. The corneal endothelium showed positivity in about 50% of cells. GrB was weakly present in healthy corneal epithelium and endothelium. The expression of both proteins was confirmed using qRT‐PCR. Besides altered expression in pathological corneas, strong positivity for PI‐9 and GrB was present in stromal infiltrates and some stromal cells that were positive for HLA‐DR and CD45. A marker typical of keratocytes, CD34, was decreased or totally absent in pathological infiltrates.Conclusions: The changes of PI‐9 and GrB expression in pathological tissue indicate that these proteins participate in the corneal immune response during the inflammatory process.

Abstract 4512: Construction and characterization of novel, human serine protease granzyme B-based cancer therapeutics targeting the TNFR family member Fn14

Abstract The TNF superfamily cytokine TWEAK and its receptor Fn14 have emerged as potentially clinically valuable targets for cancer therapy. The expression of Fn14 is comparatively low in normal tissues but elevated in various human solid tumor types, including brain, breast, lung and melanoma, and has been shown to be a negative prognostic indicator. We generated Granzyme B (GrB) containing, Fn14-targeted constructs using either the extracellular domain (ECD) of the human TWEAK ligand (GrB-TWEAK) or an engineered anti-Fn14 humanized single-chain antibody (GrB-Fc-IT4) as the targeting moieties. Both fusion constructs were expressed in mammalian HEK293T cells and purified to homogeneity from conditioned media. Both GrB-TWEAK and GrB-Fc-IT4 constructs were designed to be relatively large in molecular weight (150 kDa and 160 kDa respectively) to allow prolonged circulation in vivo. BiaCore analysis of GrB-TWEAK and GrB-Fc-IT4 binding to the Fn14 ECD indicated that these proteins bound to Fn14 with Kds of ∼8 nM and ∼18 nM, respectively. Confocal immunofluorescence analysis showed that both proteins specifically and rapidly (within 3 hrs) internalized into Fn14-expressing MDA-MB-231 breast cancer and HT-29 colorectal adenocarcinoma cells. GrB-TWEAK and GrB-Fc-IT4 demonstrated impressive and selective cytotoxicity to a panel of Fn14-expressing human tumor cell lines in the low nanomolar range. The endogenous presence of the GrB proteinase inhibitor 9 (PI-9) had no impact on the cytotoxic effect of either construct. Treatment of cells expressing the multidrug resistance protein MDR1 showed no cross-resistance to the fusion constructs in vitro. Mechanistic studies demonstrated that GrB-TWEAK and GrB-Fc-IT4 activated caspase cascades and cytochrome C-related pro-apoptotic mechanisms consistent with the known intracellular functions of GrB in target cells. Treatment of mice bearing well-established HT-29 xenografts with GrB-TWEAK (at 20 or 40 mg/kg total doses) showed significant tumor growth inhibition compared to controls (P< 0.05). GrB-TWEAK and GrB-Fc-IT4 were administered to mice bearing MDA-MB-231 orthotopic human breast xenograft tumors and both constructs displayed significant tumor growth inhibition. Treatment with GrB-Fc-IT4 displayed a more pronounced and prolonged tumor growth inhibition and longer survival than GrB-TWEAK treatment. IHC analysis demonstrates excellent tumor uptake of both agents. These data suggest that Fc-containing fusions with GrB may have a design advantage with respect to efficacy and may form the basis of novel, highly effective and nontoxic constructs for targeted therapeutic applications. Research conducted, in part, by the Clayton Foundation for Research (MGR) and National Institutes of Health grant R01 NS055126 (JAW). Citation Format: Hong Zhou, Khalid A. Mohamedali, Yu Cao, Mary Migliorini, Lawrence H. Cheung, Walter N. Hittelman, Jeffrey A. Winkles, Michael G. Rosenblum. Construction and characterization of novel, human serine protease granzyme B-based cancer therapeutics targeting the TNFR family member Fn14. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4512. doi:10.1158/1538-7445.AM2014-4512

B-CLL cells acquire APC- and CTL-like phenotypic characteristics after stimulation with CpG ODN and IL-21

CpG oligodeoxynucleotides (CpG) and IL-21 are two promising agents for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Recently, we reported that the combination of CpG and IL-21 (CpG/IL-21) can induce granzyme B (GrB)-dependent apoptosis in B-CLL cells. Here, we demonstrate that treatment of B-CLL cells with CpG and IL-21 results in the development of antigen-presenting cell (APC)-like cells with cytotoxic features. These properties eventually give rise to B-CLL cell apoptosis, independently of their cytogenetic phenotype, whereas normal B-cell survival is not negatively affected by CpG/IL-21. APC- and CTL-typical molecules found to be up-regulated in CpG/IL-21-stimulated B-CLL cells include GrB, perforin, T-bet, monokine-induced by IFN-γ and IFN-γ-inducible protein 10 (IP-10), as well as molecules important for cell adhesion, antigen cross-presentation and costimulation. Also induced are molecules involved in GrB induction, trafficking and processing, whereas the GrB inhibitor Serpin B9 [formerly proteinase inhibitor-9 (PI-9)] is down-modulated by CpG/IL-21. In conclusion, CpG/IL-21-stimulated B-CLL cells acquire features that are reminiscent of killer dendritic cells, and which result in enhanced immunogenicity, cytotoxicity and apoptosis. Our results provide novel insights into the aberrant immune state of B-CLL cells and may establish a basis for the development of an innovative cellular vaccination approach in B-CLL.

Delivery of a granzyme B inhibitor gene using carbamate-mannose modified PEI protects against cytotoxic lymphocyte killing

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells protect vertebrates by killing infected or transformed cells using granzyme B (GrB) to induce apoptosis. However, GrB-induced apoptosis of target cells causes inflammatory disease and chronic transplant rejection and so is an important disease target. The aim of this study was to prevent apoptosis of the target cells by delivering a plasmid encoding GrB inhibitor proteinase inhibitor-9 (PI-9) using cationic polymers as a non-viral vector. Polyethyleneimine (PEI, branched, Mn 10 kDa) gives a high degree of gene transfection efficiency in many types of cell lines, but it is highly cytotoxic. To reduce this cytotoxicity, we modified PEI by blocking primary amine groups through nucleophilic addition between primary amine and a protected mannose-functionalized cyclic carbonate (MTC-ipman), generating a carbamate linkage through the ring-opening of the cyclic carbonate. Deprotection of the mannose yielded a PEI polymer that is decorated with the carbohydrate. PEI with 7 or 20 of 67 primary amine groups substituted by the carbohydrate had similar gene binding ability compared to unmodified PEI, leading to almost 100% transfection efficiency of a GFP-reporter plasmid in HEK293T human embryonic kidney cells. Furthermore, modification of PEI resulted in a decrease in the cytotoxicity of PEI/DNA complexes. However, PEI with all primary amine groups blocked was unable to form a complex with DNA, and so reporter transfection was negligible. The PI-9 encoding plasmid was transfected into HEK293T cells effectively using the modified PEIs with the optimal degree of primary amine substitution, protecting up to 80% HEK293T cells from killing by human natural killer-like leukemic YT cells. Therefore, these carbamate-mannose modified PEI/PI-9 encoding plasmid complexes have potential clinical utility in the prevention of chronic transplant rejection and inflammatory disease caused by GrB.

Anti-Inflammatory Properties of Exenatide in Human Pancreatic Islets

Exenatide is an analog of the incretin hormone glucagon-like peptide (GLP-1) that is used for the treatment of T2D for their metabolic effects. In addition to its insulinotropic effects, exenatide increases functional islet mass and improves their survival. Improved outcomes have been reported in recent clinical islet transplantation trials for the treatment of type 1 diabetes. The purpose of this study was to investigate whether exenatide has anti-inflammatory properties in human islets. Exenatide treatment improved islet function, significantly reduced content of inflammation-related molecules (tissue factor, IFN-γ, IL-17, IL-1β, and IL-2) and caspase 3 activation, whereas increased phosphorylation of ERK1/2, STAT3, and Akt in vitro. Immunostaining showed expression of GLP-1R in β-cells but not in α-cells. IL-1β colocalized with GLP-1R in β-cells. Induction of serine proteinase inhibitor 9 (PI-9) was detected after exposure of human islets to exenatide in vitro and after transplantation into immunodeficient mice. GLP-1 induced PI-9 expression in vitro but to a lower extent than exenatide. This effect was partially blocked by the antagonist exendin-9 in vitro. As assessed by immunostaining PI-9 is mostly expressed in β-cells but not in α-cells. In conclusion, we describe anti-inflammatory and cytoprotective properties of exenatide in human islets. Exenatide-mediated PI-9 expression, the only known granzyme B inhibitor, unveils potential immunoregulatory properties.

Increased proteinase inhibitor-9 (PI-9) and reduced granzyme B in lung cancer: Mechanism for immune evasion?

Cytotoxic CD8+ T-cells mount immune responses to cancer via cytotoxic pathways including granzyme B. Cancer cells are also known to develop immune evasion mechanisms. We hypothesised that lung cancer cells would over-express the granzyme B-inhibitor, proteinase inhibitor-9 (PI-9) and down-regulate granzyme B expression by neighbouring CD8+ T-cells.We investigated PI-9 expression in lung cancer cell lines, and primary lung cancer cells obtained at curative lung resection from cancer patients with/without chronic obstructive pulmonary disease (COPD). Granzyme B and PI-9 expression was also determined in CD8+ T-cells from the cancer and non-cancer areas of resected lung tissue and from bronchoalveolar lavage (BAL). We then evaluated the effects of conditioned media from lung cancer cell lines on granzyme B expression and the cytotoxic activity of CD8+ T-cells.PI-9 was highly expressed in lung cancer cell lines. Increased PI-9 expression was also observed in primary cancer cells vs. epithelial cells from non-cancer tissue or bronchial brushing-derived normal primary large airway epithelial cells. Expression significantly correlated with cancer stage. Significantly reduced granzyme B was noted in CD8+ T-cells from cancer vs. non-cancer tissue. Granzyme B production by CD8+ T-cells was reduced in the presence of conditioned media from lung cancer cell lines.Our data suggest that lung cancer cells utilise their increased PI-9 expression to protect from granzyme B-mediated cytotoxicity as an immune evasion mechanism, a function that increases with lung cancer stage.

The Role of Proteinase Inhibitor 9 (PI-9) and Granzyme B in Peripheral Leukocytes for GVL and GVHD After Allogeneic Hematopoietic Stem Cell Transplantation

Abstract 1984The serpin proteinase inhibitor 9 (PI-9) protects cells from serine protease granzyme B (GrB)- and perforin (PFR)-induced cytotoxicity and apoptosis by specifically inhibiting GrB. Graft-versus-leukemia (GVL) effect and graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) are the reciprocal aspects of the established immunotherapeutic approach in hematopoietic malignancies, and are thought to be caused at least in part through GrB and PFR produced by donor-derived cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. However, GrB and PFR expressions with respect to a GVL effect, and the role of PI-9 in both GVHD and GVL, are unknown. In this study we analyzed the expression of GrB, PFR and PI-9 in acute leukemia patients to whom allogeneic HSCT was performed during remission. This study was performed under the approval of the ethical committee of Kobe University Graduate Schools of Medicine and Health Sciences, and all human samples were obtained from whom a written informed consent was obtained. We first investigated the age- and gender-related differences of expressions of PI-9 and GrB mRNA by quantitative RT-PCR in mononuclear and polymorphonuclear cells from healthy volunteers. GrB and PFR mRNAs were expressed almost exclusively in mononuclear cells that included CTLs and NK cells, while PI-9 was expressed both in mononuclear and polymorphonuclear cells. GrB and PFR expression levels were comparable among all age and gender groups. Intriguingly, however, the expression of PI-9 mRNA in lymphocytes gradually increased with age, and the PI-9 expression in the volunteer group aged 60 years old or older was significantly higher than the one in the group aged 20 to 39 years old (p=0.002). Meanwhile, the PI-9 expression in polymorphonuclear cells was comparable among all age groups, and there was no gender difference in PI-9 expression levels. Next, we analyzed the expressions of PI-9, GrB and PFR mRNAs in mononuclear and polymorphonuclear cells of 3 patients (male 2, female 1) with acute GVHD and 11 patients without acute GVHD (male 6, female 5). The GrB, PFR and PI-9 expression levels were comparable between patient groups with and without acute GVHD, both before transplantation and a week after engraftment. However, the GrB and PFR expressions prominently augmented when acute GVHD became overt in all 3 patients, while PI-9 expression level increased mildly. As a result, the ratio of GrB (or PFR) and PI-9 expressions was stable in patients with and without acute GVHD. Among them, 4 cohort patients eventually relapsed and 10 cohorts remained in remission. The GrB mRNA expression in lymphocytes one week after engraftment was 3-fold higher in 10 patients without relapse of primary disease than in 4 patients who eventually relapsed (P=0.044). This might suggest that GrB plays a role in eliciting a GVL effect as well as acute GVHD. On the other hand, PFR and PI-9 mRNA expressions were then relatively stable among patients both with and without later relapse (P=0.667; P=0.103), and the ratio of GrB (or PRF) and PI-9 expressions did not change. The expressions of GrB and PFR mRNAs in polymorphonuclear cells of all patients were under the detectable level throughout the course. Finally, we analyzed the expressions of GrB, PFR and PI-9 in leukocytes 6 months after HSCT, to see if these expressions might change in patients with chronic GVHD. Among 14 patients with chronic GVHD, the GrB expression increased (up to 6.2-fold) in 10 patients, but the PFR expression did not change. In contrast, the expression of PI-9 decreased profoundly in all of the cases both with and without chronic GVHD, relative to healthy volunteers (P=0.028). Importantly, the ratio of GrB and PI-9 expressions was significantly higher in patients with chronic GVHD than in those without GVHD (P = 0.035). In conclusion, (1) high PI-9 expression in elderly people might explain a mechanism of escape from tumor immunity in them, where PI-9 might inhibit functions of cytotoxic T cells and NK cells; (2) a high expression of GrB shortly after engraftment may be a novel biomarker for a GVL effect; and (3) a low level of PI-9 expression or an increased ratio of GrB/PI-9 later after allogeneic HSCT might be an important biomarker for cGVHD. Monitoring of both GrB and PI-9 mRNAs in peripheral blood after allogeneic HSCT could be useful for predicting the GVL effect, as well as for monitoring both acute and chronic GVHD. Disclosures:No relevant conflicts of interest to declare.

Small Molecule Inhibitors as Probes for Estrogen and Androgen Receptor Action

Because activated estrogen (ER) and androgen (AR) receptors stimulate cell proliferation in breast and prostate cancer, inhibiting their actions represents a major therapeutic goal. Most efforts to modulate ER and AR activity have focused on inhibiting the synthesis of estrogens or androgens or on the identification of small molecules that act by competing with agonist hormones for binding in the ligand-binding pocket of the receptor. An alternative approach is to implement screens for small molecule inhibitors that target other sites in the pathway of steroid receptor action. Many of these second-site inhibitors directly target ER or AR; others have still unknown sites of action. Small molecule inhibitors that target second sites represent new leads with clinical potential; they serve as novel modulators of receptor action; and they can reveal new and as yet unidentified interactions and pathways that modulate ER and AR action.

The presence and suggested role of mesothelial proteins in the human corneal endothelium

Abstract Purpose To determine whether proteins that are characteristic of the human mesothelial cell phenotype are expressed in human corneal endothelium. Methods Cadaverous human corneo‐scleral discs and pathological corneal specimens (melted and rejected corneas) were used. The detection of mesothelin, proteinase inhibitor‐9 (PI‐9), calretinin and HBME‐1 protein (a membrane protein of mesothelial cells) was performed on cryosections and corneal endothelial imprints using indirect immunofluorescent or enzymatic immuncytochemistry. The staining intensity was assessed using fluorescent or light microscopy, and the percentage of positive cells was calculated. Semi‐quantitative RT PCR of PI‐9, mesothelin and calretinin was performed using mRNA isolated from endothelial imprints. Results A strong signal for mesothelin was present in the corneal epithelium, while less intensive staining was visible in the endothelium. These results were confirmed using qRT‐PCR. Immunostaining for PI‐9 was observed in almost all corneal epithelial cells and in approximately 50% of the endothelial cells. An increased number of PI‐9‐positive cells was detected in stromal infiltrates of most melted corneal explants, while almost no positivity was observed in rejected explants. Calretinin was detected in the corneal epithelium and less intensively in the corneal endothelium, where both cytoplasmic and nuclear localisation were demonstrated. HBME‐1 antibody strongly stained the corneal endothelium and stromal keratocytes. Conclusion The proteins expressed constitutively in mesothelial cells (PI‐9, mesothelin, calretinin and HBME‐1 protein) are present in adult human corneal endothelium, confirming that this layer expresses markers typical of mesothelial cells.

Expression of proteinase inhibitor-9/serpinB9 in non-small cell lung carcinoma cells and tissues

Human proteinase inhibitor-9 (PI-9)/serpinB9 is an intracellular ovalbumin-family serpin with nucleocytoplasmic distribution which is expressed in certain normal cell types and cancer cells of different origin. Due to binding and inactivating of granzyme B (GrB), PI-9 can protect the cells from GrB-mediated apoptosis. High levels of PI-9 expression in certain cancer cells may contribute to their resistance against the immune mediated killing. So far, it is not known whether non-small cell lung cardinomas (NSCLCs) express PI-9 mRNA and a functional PI-9 protein. Herein we report for the first time that NSCLC cells express both PI-9 mRNA and protein and that there is a subset of NSCLC cells with upregulated PI-9 mRNA and protein expression. Futhermore, our work revealed that the PI-9 protein expressed in NSCLC cells can inhibit the active GrB. Finally, analysis of PI-9 mRNA expression in NSCLC tumours from surgically treated patients showed that the expression of this transcript is upregulated in the less-differentiated lung adenocarcinomas. We suggest, that the upregulated expression of PI-9 in NSCLC cells may serve to protect them from apoptosis induced by GrB.

Low Concentrations of the Soy Phytoestrogen Genistein Induce Proteinase Inhibitor 9 and Block Killing of Breast Cancer Cells by Immune Cells

The risks and benefits of diets and supplements containing the estrogenic soy isoflavone genistein are not well established. We report that 10 nm genistein potently induces the granzyme B inhibitor, proteinase inhibitor 9 (PI-9) in MCF-7 human breast cancer cells. By inducing PI-9, genistein inhibits the ability of human natural killer (NK) cells to lyse the target breast cancer cells. In ERalphaHA cells, stably transfected MCF-7 cells, which contain elevated levels of estrogen receptor-alpha (ERalpha), 100 pm genistein or 17beta-estradiol potently induce PI-9 and prevent NK cells from killing the target breast cancer cells. The concentrations of genistein that fully induce PI-9 in MCF-7 cells, and in ERalphaHA cells, are far lower than those previously reported to elicit estrogenic responses through ERalpha. Because 4-hydroxytamoxifen, raloxifene, and ICI 182,780/Faslodex all block genistein induction of PI-9 and elevated levels of ERalpha enhance induction of PI-9, genistein acts via ERalpha to induce PI-9. Increasing levels of ERalpha in breast cancer cells results in a progressive increase in induction of PI-9 by genistein and in the cell's ability to evade killing by NK cells. Moderate levels of dietary genistein and soy flour effectively induce PI-9 in human breast cancers grown in ovariectomized athymic mice. A significant population consumes levels of genistein in soy products that may be high enough to induce PI-9, perhaps potentiating the survival of some preexisting breast cancers by enabling them to evade immunosurveillance.

Non-invasive diagnosis of acute rejection in kidney transplants with delayed graft function

Delayed graft function (DGF) often occurs in kidney transplants from deceased donors. We wanted to provide studies giving more accurate non-invasive tests for acute rejection (AR). Using real-time PCR, we examined the expression of cytolytic molecules such as perforin, granzyme B, and fas-ligand along with serpin proteinase inhibitor-9. We also measured the expression of FOXP3, a characteristic gene of T-regulatory cells known to be involved in AR. These studies were conducted on peripheral blood monocytes, urinary cells, and 48 surveillance kidney biopsies taken from a total of 35 patients with DGF. Of these patients, 20 had a histopathological diagnosis of AR, whereas other 28 had characteristics of acute tubular necrosis (ATN). Expression of cytolytic and apoptotic-associated genes in the biopsy tissue, peripheral blood leukocytes, and urinary cells was significantly higher in patients with AR than that in patients with ATN. Diagnostic parameters associated with FOXP3 gene expression were most accurate in peripheral blood leukocytes and urine cells with sensitivity, specificity, positive and negative predictive values, and accuracy between 94 and 100%. Our study shows that quantification of selected genes in peripheral blood leukocytes and urinary cells from renal transplant patients with DGF may provide a useful and accurate non-invasive diagnosis of AR.

Proteinase inhibitor–9 expression is induced by maturation in dendritic cells via p38 MAP kinase

Dendritic cells (DC) are crucial for the induction of CD8(+) T cell responses. However, as DC present antigen, they may also be at risk for being killed by recent activated CD8+ T cells. Previously we have shown that lipopolysaccharide (LPS)- or CD40L-stimulated monocyte-derived DC express high levels of the granzyme B-inhibitory serpin proteinase inhibitor-9 (PI-9). Furthermore we found that its murine homolog serine protease inhibitor-6 (SPI-6) protected murine DC against cytotoxic T lymphocyte-induced apoptosis. These data suggest that PI-9/SPI-6 may regulate survival of DC when they are under attack by effector cells. We therefore analyzed how PI-9 is regulated upon stimulation with several Toll-like receptor ligands. We found that the expression of PI-9 correlated with maturation, as measured with CD86 levels, but not with the production of interleukin-12-p70. Using LPS as stimulus, we also showed that the induction of PI-9 is dependent on the p38 mitogen-activated protein kinase signaling pathway. The data suggest that PI-9 is tightly linked to maturation and may allow DC to exert their function in a potentially hostile environment.

Molecular Biology: Estrogens Shield Breast Cancer Cells

Among the more insidious aspects of cancer is its capacity for escaping the anti-cancer defenses of the host. New research suggests that some estrogens may further reinforce this evasion of host immunity, even as those same hormones stimulate the growth and spread of hormone-responsive cancers. According to David Shapiro, a medical and biochemistry professor at the University of Illinois at Urbana–Champaign, the new findings highlight the role that estrogen-related interference with immune cell function may play in the development and progression of breast cancer. Shapiro and his colleagues, reporting in a paper posted 22 January 2007 ahead of publication in Oncogene, observed that estrogen induces the expression of a newly discovered gene in humans for proteinase inhibitor 9 (PI-9). When estrogen binds to receptors in the cancer cell, the resulting complex promotes production of the PI-9 protein, which in turn binds granzyme B, the primary protease used by natural killer (NK) cells to kill off transformed or infected cells. NK cells normally play a central role in immune surveillance against the metastatic spread of cancer. Previous research out of Leiden University Medical Center in the Netherlands, published in the 25 September 2001 issue of Proceedings of the National Academy of Sciences, had shown that increased levels of PI-9 in some tumors and tumor-derived cell lines may enhance these tumors’ ability to evade apoptosis mediated by NK cells and cytotoxic T cells. In the current paper, the researchers linked increasing concentrations of estrogen with increasing levels of PI-9 and progressively blocked cell death by NK cells. “The levels of estrogen required to induce [PI-9] in breast cancer cells are extremely low,” says Shapiro. Moreover, at elevated levels of epidermal growth factor and estrogen receptor–α, the induction of PI-9 by either estradiol or the breast cancer drug tamoxifen effectively blocked the killing of cancer cells by NK cells. Levels of epidermal growth factor are elevated in many women with breast cancer, and this adversely affects the prognosis. The presence of estrogen receptor–α at the time of diagnosis is an indication for using tamoxifen or other forms of endocrine therapy. Tamoxifen is known to have mixed agonist/antagonist effects on estrogen, stimulating uterine cancer at low doses while inhibiting this and other estrogen-responsive cancers at higher doses. In contrast, the drug raloxifene, which is commonly used to prevent osteoporosis and has also been shown to reduce the risk of some breast cancers, had no immune-disrupting effects. Shapiro’s findings come with the following caveat: most estrogen-responsive breast tumors contain low to moderate levels of estrogen receptors, and in these tumors tamoxifen will not induce a level of PI-9 that enables the breast cancer cells to evade killing by immune cells. Only in the relatively small subset of breast tumors that contain very high levels of estrogen receptors will tamoxifen have an effect that could effectively shield the cancer from attacking immune cells. Indeed, a 21 June 2006 JAMA study—a prospective, double-blind, randomized clinical trial conducted in nearly 200 cancer treatment centers throughout North America—directly compared tamoxifen and raloxifene, and found both drugs to be chemopreventive. “In that large trial, tamoxifen even [seemed to perform] better in the control of noninvasive breast cancers,” says V. Craig Jordan, scientific director for the Fox Chase Cancer Center Medical Science Division. “So while these [University of Illinois] data are important and insightful, we should not lose sight of what the clinical data are telling us.” Estrogenic compounds such as DDT and dioxins may also impede immune protection against breast cancer cells. “The mechanism that this study provides for estrogen-mediated decreases in NK cell function may also be operative in the decreases in NK function that have been observed with certain environmental estrogens, such as DDT,” says Margaret Whalen, an associate professor of chemistry at Tennessee State University who has studied the immune-suppressive effects of various organochlorine pesticides. Most human exposures to xenoestrogens involve mixtures at relatively low levels, Whalen adds, and concurrent exposure to multiple chemicals may alter the immunotoxicity of a particular chemical. Shapiro and his colleagues are now looking into the factors that contribute to regulation of PI-9 in postmenopausal women. “Blocking the production of this protein represents a potential new target for breast cancer therapies,” he says.

Lithium-induced upregulation of the granzyme B inhibitor proteinase inhibitor 9 (PI-9) in monocytes: Role of glycogen synthase kinase-3? (89.13)

Abstract Proteinase inhibitor 9 (PI-9) - the only known natural antagonist of the lymphocyte protease granzyme B (GrB) - is an intracellular serpin expressed in lymphocytes and monocyte-derived cells. By intracellular flow cytometry, we have previously shown that ex-vivo stimulation by lipopolysaccharides leads to upregulation of PI-9 within 24 hours in the monocyte, but not the lymphocyte fraction; this can be inhibited by the NF-kappaB inhibitor pyrrolidin dithiocarbamate (PTDC). Glycogen synthase kinase 3 (GSK) is a lithium-inhibitable, constutitively active serine/threonine kinase that controls a number of intracellular functions, especially those involving NF-kappaB regulated genes. We now asked whether lithium-induced inhibition of GSK might influence expression of PI-9 in monocytes and lymphocytes. A 24 hours incubation assay was done using heparinized full blood of healthy volonteers, and PI-9 expression was analysed by intracellular flow cytometry. We found that lithium chloride leads to PI-9 upregulation in monocytes at a medium concentration (1.5 μM), which could not further be enhanced by higher concentrations (10 or 20 μM lithium chloride). In lymphocytes, no alteration of PI-9 expression was induced. When cells were stimulated by LPS in the presence of lithium, only marginal additional upregulation of PI-9 could be induced. Our data show that a lithium-regulated factor, probably GSK, is involved in PI-9 expression in monocytes, down-regulating PI-9 in resting cells. Since it has been shown that PI-9 over-expression in antigen presenting cells, e.g. dendritic cells, leads to an enhanced immune response, our finding may be relevant for specific immune regulation. Pharmacological manipulation of PI-9 expression may represent a useful tool to study immune regulatory mechanisms as well as for therapy .

Granzyme B leakage-induced apoptosis is a crucial mechanism of cell death in nasal-type NK/T-cell lymphoma

This study aims to investigate the role of granzyme B in the apoptosis of nasal-type NK/T-cell lymphoma. Twenty-four nasal-type NK/T-cell lymphomas were examined by TdT-mediated deoxyuridine triphosphate (dUTP)-biotin nick-end labeling (TUNEL) assay and immunohistochemical staining for active caspase 3, poly(ADP-ribose) polymerase (PARP-1/p85)/p85, and Bcl-2. In addition, HANK-1 and NKL cell lines were analyzed using Western blot analysis. Immunoprecipitation was performed to identify the binding of granzyme B and intrinsic serpin proteinase inhibitor 9 (PI-9). To localize granzyme B, immunogold labeling and immunofluorescence staining were performed. The expression level of granzyme B in tumor tissue was correlated with the apoptosis rate (P=0.015), degree of necrosis (P=0.002), and the levels of active caspase 3 (P=0.036) and poly ADP-ribose polymerase (PARP)-1/p85 (P=0.040). The granzyme B-positive HANK-1 cell line showed increased spontaneous cell death compared to the granzyme B-negative NKL cell line. The untreated HANK-1 cells released cytochrome c into the cytosol with cleavage of caspase 3 and PARP-1. Treatment with granzyme B inhibitor and caspase inhibitor decreased the cleavage of PARP-1. By performing immunogold labeling, granzyme B was identified within the cytolytic granules as well as in the cytosol. Confocal microscopy and immunoprecipitation assays confirmed the colocalization of PI-9 and granzyme B, which formed an SDS-resistant complex. These results suggested that granzyme B leakage induces cell death in NK/T-cell lymphomas via both caspase-dependent and -independent mechanisms, and this leads to the extensive necrosis that is commonly seen in NK/T-cell lymphoma.

Cloning of pig serine proteinase inhibitor 9 and its use in protecting against apoptosis

The activity of granzyme B, a main effector molecule of natural killer (NK) cells and cytotoxic T lymphocytes, is regulated by the intracellular serine proteinase inhibitor 9 (PI-9). Pig PI-9 was first cloned, and the sequences that encode pig PI-9, including the start codon and stop codon, were identified. The cDNA was inserted into the cloning site of pCXN2 (chicken beta actin promoter and cytomegalovirus enhancer), transfected into pig endothelial cells (PEC), and several stable PEC clones were established. An NK cell-mediated cytolysis test was next applied to the PEC clones, using YT cells (an NK-like cell line). The PEC transfectants with pig PI-9 had a significant inhibitory effect on NK cell-mediated PEC lysis. The overexpression of the anti-apoptotic molecule, pig PI-9, has the potential for use in protecting graft cells from human NK cells.