Serpinb9 is a marker of antigen cross-presenting dendritic cells.

Serpinb9 (Sb9, also called Spi6) is an intracellular inhibitor of granzyme B (grB) that protects cytotoxic lymphocytes from grB-mediated death. In addition, Sb9 is also expressed in accessory immune cells, including dendritic cells (DCs), although its role is debated. Recently, we have demonstrated that Sb9 plays a grB-independent role in cross-presentation of antigens by CD8+ DCs. Here, using a mouse line expressing green fluorescent protein knocked in under the control of the Sb9 promoter, we demonstrate that Sb9 expression is highest in those tissue-resident and migratory DC subsets capable of cross-presentation. Further, we show that CD8+ DCs can be divided into two subsets based on Sb9 expression, and that only the subset expressing higher levels of Sb9 is capable of cross-presentation. These findings add support for role for Sb9 cross-presentation, and indicate that high Sb9 expression is a novel marker of cross-presentation capable DCs.

NK cell intrinsic regulation of MIP-1α by granzyme M.

Granzymes are generally recognized for their capacity to induce various pathways of perforin-dependent target cell death. Within this serine protease family, Granzyme M (GrzM) is unique owing to its preferential expression in innate effectors such as natural killer (NK) cells. During Listeria monocytogenes infection, we observed markedly reduced secretion of macrophage inflammatory protein-1 alpha (MIP-1α) in livers of GrzM-deficient mice, which resulted in significantly impaired NK cell recruitment. Direct stimulation with IL-12 and IL-15 demonstrated that GrzM was required for maximal secretion of active MIP-1α. This effect was not due to reduced protein induction but resulted from heightened intracellular accumulation of MIP-1α, with reduced release. These results demonstrate that GrzM is a critical mediator of innate immunity that can regulate chemotactic networks and has an important role in the initiation of immune responses and pathogen control.

The granzyme B-Serpinb9 axis controls the fate of lymphocytes after lysosomal stress.

Cytotoxic lymphocytes (CLs) contain lysosome-related organelles (LROs) that perform the normal degradative functions of the lysosome, in addition to storage and release of powerful cytotoxins employed to kill virally infected or abnormal cells. Among these cytotoxins is granzyme B (GrB), a protease that has also been implicated in activation (restimulation)-induced cell death of natural killer (NK) and T cells, but the underlying mechanism and its regulation are unclear. Here we show that restimulation of previously activated human or mouse lymphocytes induces lysosomal membrane permeabilisation (LMP), followed by GrB release from LROs into the CL cytosol. The model lysosomal stressors sphingosine and Leu-Leu-methyl-ester, and CLs from gene-targeted mice were used to show that LMP releases GrB in both a time- and concentration-dependent manner, and that the liberated GrB is responsible for cell death. The endogenous GrB inhibitor Serpinb9 (Sb9) protects CLs against LMP-induced death but is decreasingly effective as the extent of LMP increases. We also used these model stressors to show that GrB is the major effector of LMP-mediated death in T cells, but that in NK cells additional effectors are released, making GrB redundant. We found that limited LMP and GrB release occurs constitutively in proliferating lymphocytes and in NK cells engaged with targets in vitro. In Ectromelia virus-infected lymph nodes, working NK cells lacking Sb9 are more susceptible to GrB-mediated death. Taken together, these data show that a basal level of LMP occurs in proliferating and activated lymphocytes, and is increased on restimulation. LMP releases GrB from LROs into the lymphocyte cytoplasm and its ensuing interaction with Sb9 dictates whether or not the cell survives. The GrB-Sb9 nexus may therefore represent an additional mechanism of limiting lymphocyte lifespan and populations.

A functional genomics screen identifies PCAF and ADA3 as regulators of human granzyme B-mediated apoptosis and Bid cleavage.

The human lymphocyte toxins granzyme B (hGrzB) and perforin cooperatively induce apoptosis of virus-infected or transformed cells: perforin pores enable entry of the serine protease hGrzB into the cytosol, where it processes Bid to selectively activate the intrinsic apoptosis pathway. Truncated Bid (tBid) induces Bax/Bak-dependent mitochondrial outer membrane permeability and the release of cytochrome c and Smac/Diablo. To identify cellular proteins that regulate perforin/hGrzB-mediated Bid cleavage and subsequent apoptosis, we performed a gene-knockdown (KD) screen using a lentiviral pool of short hairpin RNAs embedded within a miR30 backbone (shRNAmiR). We transduced HeLa cells with a lentiviral pool expressing shRNAmiRs that target 1213 genes known to be involved in cell death signaling and selected cells with acquired resistance to perforin/hGrzB-mediated apoptosis. Twenty-two shRNAmiRs were identified in the positive-selection screen including two, PCAF and ADA3, whose gene products are known to reside in the same epigenetic regulatory complexes. Small interfering (si)RNA-mediated gene-KD of PCAF or ADA3 also conferred resistance to perforin/hGrzB-mediated apoptosis providing independent validation of the screen results. Mechanistically, PCAF and ADA3 exerted their pro-apoptotic effect upstream of mitochondrial membrane permeabilization, as indicated by reduced cytochrome c release in PCAF-KD cells exposed to perforin/hGrzB. While overall levels of Bid were unaltered, perforin/hGrzB-mediated cleavage of Bid was reduced in PCAF-KD or ADA3-KD cells. We discovered that PCAF-KD or ADA3-KD resulted in reduced expression of PACS2, a protein implicated in Bid trafficking to mitochondria and importantly, targeted PACS2-KD phenocopied the effect of PCAF-KD or ADA3-KD. We conclude that PCAF and ADA3 regulate Bid processing via PACS2, to modulate the mitochondrial cell death pathway in response to hGrzB.

Mouse granzyme A induces a novel death with writhing morphology that is mechanistically distinct from granzyme B-induced apoptosis.

Human and mouse granzyme (Gzm)B both induce target cell apoptosis in concert with pore-forming perforin (Pfp); however the mechanisms by which other Gzms induce non-apoptotic death remain controversial and poorly characterised. We used timelapse microscopy to document, quantitatively and in real time, the death of target cells exposed to primary natural killer (NK) cells from mice deficient in key Gzms. We found that in the vast majority of cases, NK cells from wild-type mice induced classic apoptosis. However, NK cells from syngeneic Gzm B-deficient mice induced a novel form of cell death characterised by slower kinetics and a pronounced, writhing, 'worm-like' morphology. Dying cells initially contracted but did not undergo membrane blebbing, and annexin-V staining was delayed until the onset of secondary necrosis. As it is different from any cell death process previously reported, we tentatively termed this cell death 'athetosis'. Two independent lines of evidence showed this alternate form of death was due to Gzm A: first, cell death was revealed in the absence of Gzm B, but was completely lost when the NK cells were deficient in both Gzm A and B; second, the athetotic morphology was precisely reproduced when recombinant mouse Gzm A was delivered by an otherwise innocuous dose of recombinant Pfp. Gzm A-mediated athetosis did not require caspase activation, early mitochondrial disruption or generation of reactive oxygen species, but did require an intact actin cytoskeleton and was abolished by latrunculin B and mycalolide B. This work defines an authentic role for mouse Gzm A in granule-induced cell death by cytotoxic lymphocytes.

Granzyme B triggers a prolonged pressure to die in Bcl-2 overexpressing cells, defining a window of opportunity for effective treatment with ABT-737.

Overexpression of Bcl-2 contributes to resistance of cancer cells to human cytotoxic lymphocytes (CL) by blocking granzyme B (GraB)-induced mitochondrial outer membrane permeabilization (MOMP). Drugs that neutralise Bcl-2 (e.g., ABT-737) may therefore be effective adjuvants for immunotherapeutic strategies that use CL to kill cancer cells. Consistent with this we found that ABT-737 effectively restored MOMP in Bcl-2 overexpressing cells treated with GraB or natural killer cells. This effect was observed even if ABT-737 was added up to 16 h after GraB, after which the cells reset their resistant phenotype. Sensitivity to ABT-737 required initial cleavage of Bid by GraB (gctBid) but did not require ongoing GraB activity once Bid had been cleaved. This gctBid remained detectable in cells that were sensitive to ABT-737, but Bax and Bak were only activated if ABT-737 was added to the cells. These studies demonstrate that GraB generates a prolonged pro-apoptotic signal that must remain active for ABT-737 to be effective. The duration of this signal is determined by the longevity of gctBid but not activation of Bax or Bak. This defines a therapeutic window in which ABT-737 and CL synergise to cause maximum death of cancer cells that are resistant to either treatment alone, which will be essential in defining optimum treatment regimens.

Control of granzymes by serpins.

Although proteolysis mediated by granzymes has an important role in the immune response to infection or tumours, unrestrained granzyme activity may damage normal cells. In this review, we discuss the role of serpins within the immune system, as specific regulators of granzymes. The well-characterised human granzyme B-SERPINB9 interaction highlights the cytoprotective function that serpins have in safeguarding lymphocytes from granzymes that may leak from granules. We also discuss some of the pitfalls inherent in using rodent models of granzyme-serpin interactions and the ways in which our understanding of serpins can help resolve some of the current, contentious issues in granzyme biology.

The role of serpins in vertebrate immunity.

Serine proteases are important components of the immune system, playing a role in many processes including migration, phagocytosis and elimination of virally infected and cancerous cells. Members of the serpin superfamily regulate the activity of these proteases to limit tissue damage and unwarranted cell death. This review focuses on the role of intracellular (clade B) serpins in maintaining viability of both innate and adaptive immune cells.

Blocking granule-mediated death by primary human NK cells requires both protection of mitochondria and inhibition of caspase activity.

Human GraB (hGraB) preferentially induces apoptosis via Bcl-2-regulated mitochondrial damage but can also directly cleave caspases and caspase substrates in cell-free systems. How hGraB kills cells when it is delivered by cytotoxic lymphocytes (CL) and the contribution of hGraB to CL-induced death is still not clear. We show that primary human natural killer (hNK) cells, which specifically used hGraB to induce target cell death, were able to induce apoptosis of cells whose mitochondria were protected by Bcl-2. Purified hGraB also induced apoptosis of Bcl-2-overexpressing targets but only when delivered at 5- to 10-fold the concentration required to kill cells expressing endogenous Bcl-2. Caspases were critical in this process as inhibition of caspase activity permitted clonogenic survival of Bcl-2-overexpressing cells treated with hGraB or hNK cells but did not protect cells that only expressed endogenous Bcl-2. Our data therefore show that hGraB triggers caspase activation via mitochondria-dependent and mitochondria-independent mechanisms that are activated in a hierarchical manner, and that the combined effects of Bcl-2 and direct caspase inhibition can block cell death induced by hGraB and primary hNK cells.

Modulation of the granzyme B inhibitor proteinase inhibitor 9 (PI-9) by activation of lymphocytes and monocytes in vitro and by Epstein-Barr virus and bacterial infection.

Proteinase inhibitor 9 (PI-9) is an intracellular serpin expressed in lymphocytes and monocyte-derived cells. It is the only known endogenous natural antagonist of granzyme B (GrB), and its proposed function is protection of cells from misdirected GrB. We have studied the regulation of PI-9 in primary peripheral blood mononuclear cells (PBMCs) following ex-vivo stimulation, and in PBMCs from patients suffering from viral or bacterial infections. By intracellular flow cytometry, we found identical PI-9 expression in all lymphocyte subsets, lower levels in monocytes and none in granulocytes. PI-9 was stable for 48 h in the presence of cycloheximide, indicating slow protein turnover. Incubation of PBMCs with several stimuli including lipopolysaccharide (LPS) led to up-regulation in the monocyte, but not the lymphocyte fraction, within 48 h, inhibitable by the NF-kappaB inhibitor pyrrolidin dithiocarbamate (PTDC). Up-regulation of PI-9 was observed in lymphocytes and monocytes of patients with acute Epstein-Barr virus (EBV), but not bacterial infection. Preterm infants had similar PI-9 expression as adults in monocytes, but lower in lymphocytes, decreasing during bacterial infection. Taken together, our data indicate that PI-9 is rapidly up-regulated upon stimulation of monocytes, but not lymphocytes. By protecting monocytes and macrophages from misdirected GrB in the inflammatory process, PI-9 might be involved in the regulation of antigen presentation.

The granzyme B inhibitor, PI-9, is differentially expressed during placental development and up-regulated in hydatidiform moles.

The intracellular serpin Proteinase Inhibitor-9 (PI-9) is a potent inhibitor of the cytotoxic lymphocyte (CL) proteinase granzyme B, a major effector molecule used by CLs to induce target cell apoptosis. PI-9 is produced by CLs to protect against mis-directed granzyme B. However, PI-9 expression has also been reported in immune privileged tissues. In the present study, cell-specific expression of PI-9 in placental tissue of various gestational ages was examined by immunohistochemistry. PI-9 is highly expressed by the extravillous trophoblasts that have invaded the decidua, and this high expression is maintained throughout pregnancy. Similar levels were also observed in proliferative villous cytotrophoblasts. Syncytial trophoblasts generally do not produce PI-9 to a significant level until the last few weeks of pregnancy. The villous stroma contains mixed populations of PI-9 positive and negative cells throughout pregnancy, with highest expression during the second trimester. Compared to first trimester placentas, syncytial trophoblasts of partial and complete hydatidiform moles showed marked up-regulation of PI-9. Examination of choriocarcinoma cell lines also demonstrated a very high level of PI-9 is produced by these cells, which may provide protection from granzyme B-mediated apoptosis. The cell-specific expression of PI-9 in the placenta is consistent with a function in the maintenance of immune privilege, and it is proposed that up-regulated expression of PI-9 in gestational trophoblastic diseases contributes to disease pathogenesis via immune evasion.

Functional dissociation of DeltaPsim and cytochrome c release defines the contribution of mitochondria upstream of caspase activation during granzyme B-induced apoptosis.

Loss of Bid confers clonogenic survival to granzyme B-treated cells, however the exact role of Bid-induced mitochondrial damage--upstream or downstream of caspases--remains controversial. Here we show that direct cleavage of Bid by granzyme B, but not caspases, was required for granzyme B-induced apoptosis. Release of cytochrome c and SMAC, but not AIF or endonuclease G, occurred in the absence of caspase activity and correlated with the onset of apoptosis and loss of clonogenic potential. Loss of mitochondrial trans-membrane potential (DeltaPsim) was also caspase independent, however if caspase activity was blocked the mitochondria regenerated their DeltaPsim. Loss of DeltaPsim was not required for rapid granzyme B-induced apoptosis and regeneration of DeltaPsim following cytochrome c release did not confer clonogenic survival. This functional dissociation of cytochrome c and SMAC release from loss of DeltaPsim demonstrates the essential contribution of Bid upstream of caspase activation during granzyme B-induced apoptosis.

Human clade B serpins (ov-serpins) belong to a cohort of evolutionarily dispersed intracellular proteinase inhibitor clades that protect cells from promiscuous proteolysis.

Serpins are unique among the various types of active site proteinase inhibitors because they covalently trap their targets by undergoing an irreversible conformational rearrangement. Members of the serpin superfamily are present in the three major domains of life (Bacteria, Archaea and Eukarya) as well as several eukaryotic viruses. The human genome encodes for at least 35 members that segregate evolutionarily into nine (A-I) distinct clades. Most of the human serpins are secreted and circulate in the bloodstream where they reside at critical checkpoints intersecting self-perpetuating proteolytic cascades such as those of the clotting, thrombolytic and complement systems. Unlike these circulating serpins, the clade B serpins (ov-serpins) lack signal peptides and reside primarily within cells. Most of the human clade B serpins inhibit serine and/or papain-like cysteine proteinases and protect cells from exogenous and endogenous proteinase-mediated injury. Moreover, as sequencing projects expand to the genomes of other species, it has become apparent that intracellular serpins belonging to distinct phylogenic clades are also present in the three major domains of life. As some of these serpins also guard cells against the deleterious effects of promiscuous proteolytic activity, we propose that this cytoprotective function, along with similarities in structure are common features of a cohort of intracellular serpin clades from a wide variety of species.

Perforin-independent expression of granzyme B and proteinase inhibitor 9 in human testis and placenta suggests a role for granzyme B-mediated proteolysis in reproduction.

Granzyme B (graB) plays a pivotal role in cytotoxic lymphocyte granule-mediated apoptosis through cleavage of intracellular proteins in target cells. Proteinase inhibitor-9 (PI-9) is a potent inhibitor of graB and is highly expressed in cytotoxic lymphocytes. Here, we show by immunohistochemistry that PI-9 is also abundantly expressed in human testicular Sertoli cells and placental syncytial trophoblasts. Postulating that PI-9 protects these tissues from graB-producing auto- or allo-reactive cytotoxic lymphocytes, we also stained sections for graB. Unexpectedly, graB was observed in non-cytotoxic cells in both tissues. In the adult human testis, graB was present in spermatogenic cells within the seminiferous tubule, and this was verified by in-situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemical analysis of term placentae demonstrated graB in syncytial trophoblasts, and this was confirmed by RT-PCR on primary trophoblasts from term placenta. Perforin, which is co-produced with graB by activated cytotoxic lymphocytes and is required for graB release into the target cell, was not detected in either testis or placenta. We postulate that, in these organs, graB has a perforin-independent role, involving hydrolysis of extracellular matrix components. In the testis, graB may facilitate migration of developing germ cells, while in the placenta, it may contribute to extracellular matrix remodelling during parturition.

The granzyme B inhibitor, PI-9, is present in endothelial and mesothelial cells, suggesting that it protects bystander cells during immune responses.

Proteinase inhibitor 9 (PI-9) is a 42-kDa human intracellular serpin present in cytotoxic lymphocytes (CLs). PI-9 is an extremely efficient inhibitor of the pro-apoptotic CL granule proteinase granzyme B and is thought to function in the cytosol of CLs to protect against apoptosis induced by endogenously expressed or released granzyme B, particularly during target cell killing. Here we show by immunohistochemistry that PI-9 is also present in endothelial cells, in every tissue examined. Cultured endothelial cells express functional PI-9 (as assessed by binding to recombinant granzyme B) localized to the cytoplasm and nucleus. Immunohistochemistry also showed PI-9 in mesothelial cells, and this was confirmed by analysis of primary cells cultured from pleural and serous effusions. Granzyme B expression was not detected in either endothelial or mesothelial cells. In both cell types, PI-9 is up-regulated at the mRNA and protein level by exposure to the phorbol ester PMA, consistent with a response to inflammatory stimuli. We postulate that PI-9 is present in these lining cell types to protect against misdirected, free granzyme B released during a local immune response.

Nucleocytoplasmic distribution of the ovalbumin serpin PI-9 requires a nonconventional nuclear import pathway and the export factor Crm1.

Proteinase inhibitor 9 (PI-9) is a human serpin present in the cytoplasm of cytotoxic lymphocytes and epithelial cells. It inhibits the cytotoxic lymphocyte granule proteinase granzyme B (graB) and is thought to protect cytotoxic lymphocytes and bystander cells from graB-mediated apoptosis. Following uptake into cells, graB promotes DNA degradation, rapidly translocating to the nucleus, where it binds a nuclear component. PI-9 should therefore be found in cytotoxic lymphocyte and bystander cell nuclei to ensure complete protection against graB. Here we demonstrate by microscopy and subcellular fractionation experiments that PI-9 is present in the nuclei of human cytotoxic cells, endothelial cells, and epithelial cells. We also show that the related serpins, PI-6, monocyte neutrophil elastase inhibitor (MNEI), PI-8, plasminogen activator inhibitor 2 (PAI-2), and the viral serpin CrmA exhibit similar nucleocytoplasmic distributions. Because these serpins lack classical nuclear localization signals and are small enough to diffuse through nuclear pores, we investigated whether import occurs actively or passively. Large (approximately 70 kDa) chimeric proteins comprising PI-9, PI-6, PI-8, MNEI, or PAI-2 fused to green fluorescent protein (GFP) show similar nucleocytoplasmic distributions to the parent proteins, indicating that nuclear import is active. By contrast, CrmA-GFP is excluded from nuclei, indicating that CrmA is not actively imported. In vitro nuclear transport assays show that PI-9 accumulates at a rate above that of passive diffusion, that it requires cytosolic factors but not ATP, and that it does not bind an intranuclear component. Furthermore, PI-9 is exported from nuclei via a leptomycin B-sensitive pathway, implying involvement of the export factor Crm1p. We conclude that the nucleocytoplasmic distribution of PI-9 and related serpins involves a nonconventional nuclear import pathway and Crm1p.

Sequence, organization, chromosomal localization, and alternative splicing of the human serine protease inhibitor gene hurpin (PI13) which is upregulated in psoriasis.

Hurpin (protease inhibitor 13; PI13) is the most recently identified member of the ovalbumin family of serine protease inhibitors (serpins). It is expressed in human epidermal keratinocytes and is downregulated by exposure to ultraviolet irradiation. A role for hurpin in the proliferation or differentiation of keratinocytes has been proposed because of its strong expression in proliferating cells and its deregulated expression in the lesional epidermis of psoriatic patients. Here, we report the cloning, chromosomal localization, and complete sequence of the human hurpin gene. By PCR-based screening of the GeneBridge 4 radiation hybrid panel, we mapped the gene to chromosome 18q21.3, close to a known cluster of ov-serpin genes. Using the full-length cDNA for hurpin, we identified two clones from an arrayed genomic P1 placental library that contain the entire hurpin gene. Sequencing revealed that the gene covers 12.253 kb and is comprised of eight exons and seven introns. The exon--intron boundaries are identical in position and phasing to those in other members of the 18q serpin gene cluster, and analysis of hurpin variants indicated that modified functional inhibitors, differing only in the CD interhelical loop, can be generated by differential splicing of exon 3. These data show that hurpin is a typical member of the 18q ovalbumin-serpins most closely related to the serpins squamous-cell carcinoma antigens 1 and 2.

Importance of the P4' residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop.

The cytotoxic lymphocyte serine proteinase granzyme B induces apoptosis of abnormal cells by cleaving intracellular proteins at sites similar to those cleaved by caspases. Understanding the substrate specificity of granzyme B will help to identify natural targets and develop better inhibitors or substrates. Here we have used the interaction of human granzyme B with a cognate serpin, proteinase inhibitor 9 (PI-9), to examine its substrate sequence requirements. Cleavage and sequencing experiments demonstrated that Glu(340) is the P1 residue in the PI-9 RCL, consistent with the preference of granzyme B for acidic P1 residues. Ala-scanning mutagenesis demonstrated that the P4-P4' region of the PI-9 RCL is important for interaction with granzyme B, and that the P4' residue (Glu(344)) is required for efficient serpin-proteinase binding. Peptide substrates based on the P4-P4' PI-9 RCL sequence and containing either P1 Glu or P1 Asp were cleaved by granzyme B (k(cat)/K(m) 9.5 x 10(3) and 1.2 x 10(5) s(-1) M(-1), respectively) but were not recognized by caspases. A substrate containing P1 Asp but lacking P4' Glu was cleaved less efficiently (k(cat)/K(m) 5.3 x 10(4) s(-1) M(-1)). An idealized substrate comprising the previously described optimal P4-P1 sequence (Ile-Glu-Pro-Asp) fused to the PI-9 P1'-P4' sequence was efficiently cleaved by granzyme B (k(cat)/K(m) 7.5 x 10(5) s(-1) M(-1)) and was also recognized by caspases. This contrasts with the literature value for a tetrapeptide comprising the same P4-P1 sequence (k(cat)/K(m) 6.7 x 10(4) s(-1) M(-1)) and confirms that P' residues promote efficient interaction of granzyme B with substrates. Finally, molecular modeling predicted that PI-9 Glu(344) forms a salt bridge with Lys(27) of granzyme B, and we showed that a K27A mutant of granzyme B binds less efficiently to PI-9 and to substrates containing a P4' Glu. We conclude that granzyme B requires an extended substrate sequence for specific and efficient binding and propose that an acidic P4' substrate residue allows discrimination between early (high affinity) and late (lower affinity) targets during the induction of apoptosis.