Interleukin-18 and cytotoxic impairment are independent and synergistic causes of murine virus-induced hyperinflammation.

Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening hyperinflammatory syndromes typically associated with underlying hematologic and rheumatic diseases, respectively. Familial HLH is associated with genetic cytotoxic impairment and thereby to excessive antigen presentation. Extreme elevation of serum interleukin-18 (IL-18) has been observed specifically in patients with MAS, making it a promising therapeutic target, but how IL-18 promotes hyperinflammation remains unknown. In an adjuvant-induced MAS model, excess IL-18 promoted immunopathology, whereas perforin deficiency had no effect. To determine the effects of excess IL-18 on virus-induced immunopathology, we infected Il18-transgenic (Il18tg) mice with lymphocytic choriomeningitis virus (LCMV; strain Armstrong). LCMV infection is self-limited in wild-type mice, but Prf1-/- mice develop prolonged viremia and fatal HLH. LCMV-infected Il18-transgenic (Il18tg) mice developed cachexia and hyperinflammation comparable to Prf1-/- mice, albeit with minimal mortality. Like Prf1-/- mice, immunopathology was largely rescued by CD8 depletion or interferon-γ (IFNg) blockade. Unlike Prf1-/- mice, they showed normal target cell killing and normal clearance of viral RNA and antigens. Rather than impairing cytotoxicity, excess IL-18 acted on T lymphocytes to amplify their inflammatory responses. Surprisingly, combined perforin deficiency and transgenic IL-18 production caused spontaneous hyperinflammation specifically characterized by CD8 T-cell expansion and improved by IFNg blockade. Even Il18tg;Prf1-haplosufficient mice demonstrated hyperinflammatory features. Thus, excess IL-18 promotes hyperinflammation via an autoinflammatory mechanism distinct from, and synergistic with, cytotoxic impairment. These data establish IL-18 as a potent, independent, and modifiable driver of life-threatening innate and adaptive hyperinflammation and support the rationale for an IL-18-driven subclass of hyperinflammation.

Impaired immune surveillance accelerates accumulation of senescent cells and aging.

Cellular senescence is a stress response that imposes stable cell-cycle arrest in damaged cells, preventing their propagation in tissues. However, senescent cells accumulate in tissues in advanced age, where they might promote tissue degeneration and malignant transformation. The extent of immune-system involvement in regulating age-related accumulation of senescent cells, and its consequences, are unknown. Here we show that Prf1-/- mice with impaired cell cytotoxicity exhibit both higher senescent-cell tissue burden and chronic inflammation. They suffer from multiple age-related disorders and lower survival. Strikingly, pharmacological elimination of senescent-cells by ABT-737 partially alleviates accelerated aging phenotype in these mice. In LMNA+/G609G progeroid mice, impaired cell cytotoxicity further promotes senescent-cell accumulation and shortens lifespan. ABT-737 administration during the second half of life of these progeroid mice abrogates senescence signature and increases median survival. Our findings shed new light on mechanisms governing senescent-cell presence in aging, and could motivate new strategies for regenerative medicine.

Cholangiocarcinoma-derived exosomes inhibit the antitumor activity of cytokine-induced killer cells by down-regulating the secretion of tumor necrosis factor-α and perforin.

OBJECTIVE: The aim of our study is to observe the impact of cholangiocarcinoma-derived exosomes on the antitumor activities of cytokine-induced killer (CIK) cells and then demonstrate the appropriate mechanism. METHODS: Tumor-derived exosomes (TEXs), which are derived from RBE cells (human cholangiocarcinoma line), were collected by ultracentrifugation. CIK cells induced from peripheral blood were stimulated by TEXs. Fluorescence-activated cell sorting (FACS) was performed to determine the phenotypes of TEX-CIK and N-CIK (normal CIK) cells. The concentrations of tumor necrosis factor-α (TNF-α) and perforin in the culture medium supernatant were examined by using an enzyme-linked immunosorbent assay (ELISA) kit. A CCK-8 kit was used to evaluate the cytotoxic activity of the CIK cells to the RBE cell line. RESULTS: The concentrations of TNF-α and perforin of the group TEX-CIK were 138.61 pg/ml and 2.41 ng/ml, respectively, lower than those of the group N-CIK 194.08 pg/ml (P<0.01) and 3.39 ng/ml (P<0.05). The killing rate of the group TEX-CIK was 33.35%, lower than that of the group N-CIK (47.35% (P<0.01)). The population of CD3(+), CD8(+), NK (CD56(+)), and CD3(+)CD56(+) cells decreased in the TEX-CIK group ((63.2±6.8)%, (2.5±1.0)%, (0.53±0.49)%, (0.45±0.42)%) compared with the N-CIK group ((90.3±7.3)%, (65.7±3.3)%, (4.2±1.2)%, (15.2±2.7)%), P<0.01. CONCLUSIONS: Our results suggest that RBE cells-derived exosomes inhibit the antitumor activity of CIK cells by down-regulating the population of CD3(+), CD8(+), NK (CD56(+)), and CD3(+)CD56(+) cells and the secretion of TNF-α and perforin. TEX may play an important role in cholangiocarcinoma immune escape.

Perforin: a key pore-forming protein for immune control of viruses and cancer.

Perforin (PFN) is the key pore-forming molecule in the cytotoxic granules of immune killer cells. Expressed only in killer cells, PFN is the rate-limiting molecule for cytotoxic function, delivering the death-inducing granule serine proteases (granzymes) into target cells marked for immune elimination. In this chapter we describe our current understanding of how PFN accomplishes this task. We discuss where PFN is expressed and how its expression is regulated, the biogenesis and storage of PFN in killer cells and how they are protected from potential damage, how it is released, how it delivers Granzymes into target cells and the consequences of PFN deficiency.

Perforin and human diseases.

Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) use a highly toxic pore-forming protein perforin (PFN) to destroy cells infected with intracellular pathogens and cells with pre-cancerous transformations. However, mutations of PFN and defects in its expression can cause an abnormal function of the immune system and difficulties in elimination of altered cells. As discussed in this chapter, deficiency of PFN due to the mutations of its gene, PFN1, can be associated with malignancies and severe immune disorders such as familial hemophagocytic lymphohistiocytosis (FHL) and macrophage activation syndrome. On the other hand, overactivity of PFN can turn the immune system against autologous cells resulting in other diseases such as systemic lupus erythematosus, polymyositis, rheumatoid arthritis and cutaneous inflammation. PFN also has a crucial role in the cellular rejection of solid organ allografts and destruction of pancreatic ß-cells resulting in type 1 diabetes. These facts highlight the importance of understanding the biochemical characteristics of PFN.

The role of MACPF proteins in the biology of malaria and other apicomplexan parasites.

Apicomplexans are eukaryotic parasites of major medical and veterinary importance. They have complex life cycles through frequently more than one host, interact with many cell types in their hosts, and can breach host cell membranes during parasite traversal of, or egress from, host cells. Some of these parasites make a strikingly heavy use of the pore-forming MACPF domain, and encode up to 10 different MACPF domain-containing proteins. In this chapter, we focus on the two most studied and medically important apicomplexans, Plasmodium and Toxoplasma, and describe the known functions of their MACPF polypeptide arsenal. Apicomplexan MACPF proteins appear to be involved in a variety of membrane-damaging events, making them an attractive model to dissect the structure-function relationships of the MACPF domain.

Chlamydial MACPF protein CT153.

Chlamydiae are obligate intracellular bacterial parasites that infect a wide range of metazoan hosts. Some Chlamydia species are important causes of chronic inflammatory diseases of the ocular, genital and respiratory tracts in humans. Genes located in a variable region of chlamydial genomes termed the plasticity zone are known to be key determinants of pathogenic diversity. The plasticity zone protein CT153, present only in select species, contains a membrane attack complex/perforin (MACPF) domain, which may mediate chlamydial interactions with the host cell. CT153 is present throughout the C. trachomatis developmental cycle and is processed into polypeptides that interact with membranes differently than does the parent protein. Chlamydiae interact extensively with membranes from the time of invasion until they eventually exit host cells, so numerous roles for a MACPF protein in pathogenesis of these pathogens are conceivable. Here, we present an overview of what is known about CT153 and highlight potential roles of a MACPF family protein in a group of pathogens whose intracellular development is marked by a series of interactions with host cell membranes and organelles. Finally, we identify new strategies for identifying CT153 functions made feasible by the recent development of a basic toolset for genetic manipulation of chlamydiae.

Perforin- and granulysin-mediated cytotoxicity and interleukin 15 play roles in neurocognitive impairment in patients with acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) is an aggressive disease. The course of disease is regulated by pro-inflammatory agents, and malignant cell infiltration of tissues plays a deleterious role in disease progression, greatly impacting quality of life, especially in the cognitive domains. Our hypothesis is that significant serum concentrations of interleukin 15 (IL-15) are responsible for higher expression of adhesion molecules on endothelial cells of blood-brain barrier (BBB) which allow leukaemia cells and/or normal lymphocytes the infiltration into the brain. In brain tissue these cells could be stimulated to release perforin and granulysin causing induction of apoptosis in brain cells that are involved in complex neural signalling mediated by neurotransmitters, and consequent fine cognitive impairment. Such changes could be detected early, even before notable clinical psycho-neurological or radiological changes in patients with ALL. To evaluate this hypothesis we propose measuring cognitive function using Complex Reactiometer Drenovac (CRD) scores in patients with ALL. The expression of different adhesion molecules on BBB as well as presence and distribution of different lymphocytes in brain tissue will be analyzed. We will then correlate CRD scores with levels of IL-15 and the percentages of T cells, natural killer T cells, and natural killer cells expressing perforin and/or granulysin proteins. CRD is a scientifically recognised and highly sensitive psychometric laboratory test based on the complex chronometric mathematical measuring of speed of reaction to various stimuli. It provides an objective assessment of cognitive functions from the most complex mental activities to the simplest reaction reflexes. Early recognition of cognitive dysfunction might be important when selecting the most appropriate chemotherapy and/or radiotherapy regimens, and could allow for the implementation of preventive measures against further deterioration in cognitive function and quality of life in patients with ALL.

Perforin deficiency impairs a critical immunoregulatory loop involving murine CD8(+) T cells and dendritic cells.

Humans and mice with impaired perforin-dependent cytotoxic function may develop excessive T-cell activation and the fatal disorder hemophagocytic lymphohistiocytosis (HLH) after infection. Though cytotoxic lymphocytes can kill antigen-presenting cells, the physiological mechanism of perforin-mediated immune regulation has never been demonstrated in a disease-relevant context. We used a murine model of HLH to examine how perforin controls immune activation, and we have defined a feedback loop that is critical for immune homeostasis. This endogenous feedback loop involves perforin-dependent elimination of rare, antigen-presenting dendritic cells (DCs) by CD8(+) T cells and has a dominant influence on the magnitude of T-cell activation after viral infection. Antigen presentation by a minor fraction of DCs persisted in T-cell- or perforin-deficient animals and continued to drive T-cell activation well beyond initial priming in the latter animals. Depletion of DCs or transfer of perforin-sufficient T cells dampened endogenous DC antigen presentation and T-cell activation, demonstrating a reciprocal relationship between perforin in CD8(+) T cells and DC function. Thus, selective cytotoxic "pruning" of DC populations by CD8(+) T cells limits T-cell activation and protects against the development of HLH and potentially other immunopathological conditions.

Controversies in granzyme biology.

Granzymes (Grz) are a family of serine proteases found in the granules of cytotoxic lymphocytes and are emerging as an important group of proteins involved in immune function and surveillance. Grz have both cytotoxic and more recently reported non-cytotoxic roles, however these functions are still subject to thorough investigation. The significance of the cytotoxic and importantly the non-cytotoxic roles of Grz will be discussed in this review, detailing accepted and controversial functions.

A possible role for perforin and granzyme B in resveratrol-enhanced radiosensitivity of prostate cancer.

Perforin and granzyme B are expressed primarily by activated lymphocytes (cytotoxic T cells, natural killer cells, and natural killer T cells) and function together to induce apoptosis of target cells. Typically, these proteins are not expressed in tumor cells. In the present study, we established the constitutive expression of perforin and granzyme B by the PC-3 and DU145 prostate cancer (PCA) cell lines with reverse transcription polymerase chain reaction, immunohistochemistry, Western blot, or a combination of techniques. The combination of radiation and resveratrol (XRT/RSV) additively/synergistically decreased survival of PCA because, at least in part, of increased apoptosis. We further demonstrated that treatment with RSV up-regulated the expression of both perforin and granzyme B, whereas treatment with XRT up-regulated the expression of granzyme B, but not that of perforin. Combined XRT/RSV treatment of PCA cells further increased the expression of both perforin and granzyme B compared with RSV or XRT alone. Thus, increased radiosensitivity of prostate cancer cells induced by RSV correlated with up-regulation of perforin and granzyme B, demonstrating a possible mechanism for tumor apoptosis. These findings might be helpful in devising new strategies for treating PCA.

Critical role for perforin and Fas-dependent killing of dendritic cells in the control of inflammation.

After stimulation of antigen-specific T cells, dendritic cell (DCs) are susceptible to killing by these activated T cells that involve perforin and Fas-dependent mechanisms. Fas-dependent DC apoptosis has been shown to limit DC accumulation and prevent the development of autoimmunity. However, a role for perforin in the maintenance of DC homeostasis for immune regulation remains to be determined. Here we show that perforin deficiency in mice, together with the deletion of Fas in DCs (perforin(-/-)DC-Fas(-/-)), led to DC accumulation, uncontrolled T-cell activation, and IFN-γ production by CD8+ T cells, resulting in the development of lethal hemophagocytic lymphohistiocytosis. Consistently, adoptive transfer of Fas(-/-) DCs induced over-activation and IFN-γ production in perforin(-/-) CD8+ T cells. Neutralization of IFN-γ prevented the spreading of inflammatory responses to different cell types and protected the survival of perforin(-/-)DC-Fas(-/-) mice. Our data suggest that perforin and Fas synergize in the maintenance of DC homeostasis to limit T cell activation, and prevent the initiation of an inflammatory cascade.

Granzyme B as a novel factor involved in cardiovascular diseases.

Apoptosis plays an important role in cardiovascular diseases such as atherosclerosis, ischemic heart disease, and congestive heart failure. Previous studies have demonstrated that oxidative stress, physiological stress, and inflammatory cytokines such as tumor necrosis factor and Fas ligand are involved in apoptosis of cardiovascular system. We demonstrate that another apoptosis-related pathway, i.e. granzyme B/perforin system is involved in cardiovascular diseases. Expression of granzyme B, a member of serine protease family is increased in acute coronary syndrome, coronary artery disease with end-stage renal disease, and subacute stage of acute myocardial infarction. Although granzyme B is extensively researched in immunological disorders, the role of granzyme B/perforin system was not clear in the cardiovascular field. In addition, little is known regarding the inhibition of granzyme B system in the clinical situation. In this review we demonstrate recent findings of granzyme B in cardiovascular diseases and possible therapeutic applications of inhibiting the granzyme B/perforin system.

Distinct roles of cytolytic effector molecules for antigen-restricted killing by CTL in vivo.

Cytotoxic T lymphocytes (CTLs) represent one of the front lines of defense for the immune system, killing virus-infected and tumor-transformed cells. CTL use at least two mechanisms to induce apoptosis in their targets, one mediated by perforin and granzymes, and the other triggered by the death ligand, CD95 ligand (CD95L). Here, we used an in vivo cytotoxicity assay to measure specific clearance of antigen-bearing target cells in mice that had previously been immunized with noninfectious cell-associated antigens. We found that perforin was dispensable for efficient clearance of antigen-bearing cells from immunized mice, but only if CD95/CD95L was functional; however, there was a delay in target cell clearance in the absence of perforin. In addition, we observed ∼35% target cell clearance in the absence of both perforin and CD95L, which was only slightly abrogated in the presence of a neutralizing anti-tumor necrosis factor (TNF) antibody. The presence of a dominant negative Fas-associated death domain (FADD) did not block target cell clearance and therefore cannot be attributed to known death receptors. Taken together, these data suggest that perforin- and CD95L-dependent killing are complementary at early time points, each can compensate for the absence of the other at later time points, and that there is an additional component of antigen-restricted CTL killing independent of perforin, CD95L, and TNFα.

[The role of perforin mediated cell cytotoxicity in psoriasis]. / Uloga stanicne citotoksicnosti posredovane perforinom u psorijazi.

Psoriasis is a common chronic inflammatory skin disease characterized by hyperproliferation and incomplete differentiation of epidermal keratinocytes as well as by inflammatory infiltrate of T-lymphocytes in dermis and epidermis. Psoriasis is nowadays also recognized as a T cell mediated disease resulting from aberrant activation of both innate and adaptive immunity. The main effector cells in mediating psoriatic phenotype are helper CD4+ T cells and cytotoxic CD8+ T cells. Both, CD4+ and CD8+ T cells, mediate apoptosis via the release of cell granules, perforin and granzymes or by binding of ligands to their death receptors on target cells. The role of cell cytotoxicity mechanisms, particularly those mediated by perforin, in psoriasis is as yet unclear. Perforin is a pore forming molecule, located within the cytoplasm of cytotoxic T cells and natural killer cells, which enables entry of granzymes and other apoptotic molecules into the target cell in order to mediate programmed cell death. The importance of perforin-mediated cytotoxicity has been demonstrated in several autoimmune diseases and in some inflammatory skin diseases. Recent studies claimed its role in the immunopathogenesis of psoriasis as well. Accumulation of perforin-positive cells in psoriatic epidermis close to damaged keratinocytes suggests that T lymphocytes induce damage to keratinocytes by releasing cytolytic molecules. On the other hand, apoptotic keratinocytes might trigger an injury response program causing regenerative hyperplasia of epidermal keratinocytes, a hallmark of psoriasis. Progress in understanding of effector part of cell cytotoxicity in psoriatic plaque might in future enable more specific treatment of psoriatic patients by blocking selectively each of proposed cytolytic mechanisms and molecules as potential new therapeutic targets.

Vibrio cholerae cytolysin causes an inflammatory response in human intestinal epithelial cells that is modulated by the PrtV protease.

BACKGROUND: Vibrio cholerae is the causal intestinal pathogen of the diarrheal disease cholera. It secretes the protease PrtV, which protects the bacterium from invertebrate predators but reduces the ability of Vibrio-secreted factor(s) to induce interleukin-8 (IL-8) production by human intestinal epithelial cells. The aim was to identify the secreted component(s) of V. cholerae that induces an epithelial inflammatory response and to define whether it is a substrate for PrtV. METHODOLOGY/PRINCIPAL FINDINGS: Culture supernatants of wild type V. cholerae O1 strain C6706, its derivatives and pure V. cholerae cytolysin (VCC) were analyzed for the capacity to induce changes in cytokine mRNA expression levels, IL-8 and tumor necrosis factor-alpha (TNF-alpha) secretion, permeability and cell viability when added to the apical side of polarized tight monolayer T84 cells used as an in vitro model for human intestinal epithelium. Culture supernatants were also analyzed for hemolytic activity and for the presence of PrtV and VCC by immunoblot analysis. CONCLUSIONS/SIGNIFICANCE: We suggest that VCC is capable of causing an inflammatory response characterized by increased permeability and production of IL-8 and TNF-alpha in tight monolayers. Pure VCC at a concentration of 160 ng/ml caused an inflammatory response that reached the magnitude of that caused by Vibrio-secreted factors, while higher concentrations caused epithelial cell death. The inflammatory response was totally abolished by treatment with PrtV. The findings suggest that low doses of VCC initiate a local immune defense reaction while high doses lead to intestinal epithelial lesions. Furthermore, VCC is indeed a substrate for PrtV and PrtV seems to execute an environment-dependent modulation of the activity of VCC that may be the cause of V. cholerae reactogenicity.

The roles of interferon-gamma and perforin in antiviral immunity in mice that differ in genetically determined NK-cell-mediated antiviral activity.

The design of effective antiviral immunotherapies depends on a detailed understanding of the cellular and molecular processes involved in generating and maintaining immune responses. Control of cytomegalovirus (CMV) infection requires the concerted activities of both innate and adaptive immune effectors. In the mouse, immunity to acute murine CMV (MCMV) infection depends on natural killer (NK) cells and/or CD8(+) T cells. The relative importance of NK and CD8(+) T cells varies in different mouse strains. In C57BL/6 mice, early viral infection is controlled by Ly49H(+) NK cells, whereas in BALB/c mice, CD8(+) T cells exert the principal antiviral activities. Although the role of NK and CD8(+) T cells is defined, the molecular mechanisms they utilize to limit acute infection are poorly understood. Here, we define the specific roles of perforin (pfp) and interferon-gamma (IFN-gamma) in the context of NK- or T-cell-mediated immunity to MCMV during acute infection. We show that pfp is essential for both NK- and T-cell-mediated antiviral immunity during the early stages of infection. The relative importance of IFN-gamma is more pronounced in Ly49H(-) mice. Using BALB/c background mice congenic for Ly49H and lacking pfp, we show that Ly49H-regulated NK-cell control of MCMV infection is dependent on pfp-mediated cytolysis.

Temperature sensitivity of human perforin mutants unmasks subtotal loss of cytotoxicity, delayed FHL, and a predisposition to cancer.

The pore-forming protein perforin is critical for defense against many human pathogens and for preventing a catastrophic collapse of immune homeostasis, manifested in infancy as Type 2 familial hemophagocytic lymphohistiocytosis (FHL). However, no evidence has yet linked defective perforin cytotoxicity with cancer susceptibility in humans. Here, we examined perforin function in every patient reported in the literature who lived to at least 10 years of age without developing FHL despite inheriting mutations in both of their perforin (PRF1) alleles. Our analysis showed that almost 50% of these patients developed at least 1 hematological malignancy in childhood or adolescence. The broad range of pathologies argued strongly against a common environmental or viral cause for the extraordinary cancer incidence. Functionally, what distinguished these patients was their inheritance of PRF1 alleles encoding temperature-sensitive missense mutations. By contrast, truly null missense mutations with no rescue at the permissive temperature were associated with the more common severe presentation with FHL in early infancy. Our study provides the first mechanistic evidence for a link between defective perforin-mediated cytotoxicity and cancer susceptibility in humans and establishes the paradigm that temperature sensitivity of perforin function is a predictor of FHL severity.

Altered effector function of peripheral cytotoxic cells in COPD.

BACKGROUND: There is mounting evidence that perforin and granzymes are important mediators in the lung destruction seen in COPD. We investigated the characteristics of the three main perforin and granzyme containing peripheral cells, namely CD8+ T lymphocytes, natural killer (NK; CD56+CD3-) cells and NKT-like (CD56+CD3+) cells. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated and cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD8+ T lymphocytes, NK (CD56+CD3-) and NKT-like (CD56+CD3+) cells were used in an LDH release assay to determine cytotoxicity and cytotoxic mechanisms were investigated by blocking perforin and granzyme B with relevant antibodies. RESULTS: The proportion of peripheral blood NKT-like (CD56+CD3+) cells in smokers with COPD (COPD subjects) was significantly lower (0.6%) than in healthy smokers (smokers) (2.8%, p < 0.001) and non-smoking healthy participants (HNS) (3.3%, p < 0.001). NK (CD56+CD3-) cells from COPD subjects were significantly less cytotoxic than in smokers (16.8% vs 51.9% specific lysis, p < 0.001) as were NKT-like (CD56+CD3+) cells (16.7% vs 52.4% specific lysis, p < 0.001). Both cell types had lower proportions expressing both perforin and granzyme B. Blocking the action of perforin and granzyme B reduced the cytotoxic activity of NK (CD56+CD3-) and NKT-like (CD56+CD3+) cells from smokers and HNS. CONCLUSION: In this study, we show that the relative numbers of peripheral blood NK (CD56+CD3-) and NKT-like (CD56+CD3+) cells in COPD subjects are reduced and that their cytotoxic effector function is defective.