KLF13 regulates the differentiation-dependent human papillomavirus life cycle in keratinocytes through STAT5 and IL-8.

High-risk strains of human papillomavirus (HPV) are the causative agents of cervical and anogenital cancers and are associated with 5% of all human cancers. Although prophylactic vaccines targeting a subset of HPV types are available, they are ineffective in HPV-infected individuals. Elucidation of the mechanisms controlling HPV replication may allow development of novel anti-HPV therapeutics. Infectious HPV virions are produced during terminal differentiation of host cells. The process of viral maturation requires synergistic interactions between viral and cellular proteins that leads to amplification of the viral genome and expression of late viral genes. Here we show that the transcription factor Kruppel-like factor 13 (KLF13) has a critical role in the HPV life cycle. KLF13 is overexpressed in HPV-positive keratinocytes and cervical cancer cell lines. Expression of KLF13 in normal cervical epithelium is low but increases significantly in cervical intraepithelial neoplasia and invasive squamous cervical cancer. After HPV infection, the E7 protein suppresses ubiquitin ligase FBW7 expression leading to an increase in KLF13 expression. Reduction of KLF13 with short hairpin RNA in differentiating HPV-positive cells resulted in diminished levels of viral gene expression and genome amplification. Knockdown of KLF13 also reduced the level of the transcription factor signal transducer and activator of transcription 5, which led to the downregulation of the ataxia-telangiectasia mutated DNA damage pathway and the chemokine interleukin-8 (IL-8). In addition, neutralization of IL-8 diminished viral genome amplification in differentiating HPV-positive cells. Thus, KLF13 is critical for the activation of the HPV productive life cycle and is likely involved in initiation and progression of cervical cancer.

Biology and clinical relevance of granulysin.

Granulysin is a cytolytic and proinflammatory molecule first identified by a screen for genes expressed 'late' (3-5 days) after activation of human peripheral blood mononuclear cells. Granulysin is present in cytolytic granules of cytotoxic T lymphocytes and natural killer cells. Granulysin is made in a 15-kDa form that is cleaved into a 9-kDa form at both the amino and the carboxy termini. The 15-kDa form is constitutively secreted, and its function remains poorly understood. The 9-kDa form is released by receptor-mediated granule exocytosis. Nine kiloDalton granulysin is broadly cytolytic against tumors and microbes, including gram-positive and gram-negative bacteria, fungi/yeast and parasites. It kills the causative agents of both tuberculosis and malaria. Granulysin is also a chemoattractant for T lymphocytes, monocytes and other inflammatory cells and activates the expression of a number of cytokines, including regulated upon activation T cell expressed and secreted (RANTES), monocyte chemoattractant protein (MCP)-1, MCP-3, macrophage inflammatory protein (MIP)-1 alpha, interleukin (IL)-10, IL-1, IL-6 and interferon (IFN)-alpha. Granulysin is implicated in a myriad of diseases including infection, cancer, transplantation, autoimmunity, skin and reproductive maladies. Small synthetic forms of granulysin are being developed as novel antibiotics. Studies of the full-length forms may give rise to new diagnostics and therapeutics for use in a wide variety of diseases.

Visiting a forest, but not a city, increases human natural killer activity and expression of anti-cancer proteins.

We previously reported that a forest bathing trip enhanced human NK activity, number of NK cells, and intracellular anti-cancer proteins in lymphocytes. In the present study, we investigated how long the increased NK activity lasts and compared the effect of a forest bathing trip on NK activity with a trip to places in a city without forests. Twelve healthy male subjects, age 35-56 years, were selected with informed consent. The subjects experienced a three-day/two-night trip to forest fields and to a city, in which activity levels during both trips were matched. On day 1, subjects walked for two hours in the afternoon in a forest field; and on day 2, they walked for two hours in the morning and afternoon, respectively, in two different forest fields; and on day 3, the subjects finished the trip and returned to Tokyo after drawing blood samples and completing the questionnaire. Blood and urine were sampled on the second and third days during the trips, and on days 7 and 30 after the trip, and NK activity, numbers of NK and T cells, and granulysin, perforin, and granzymes A/B-expressing lymphocytes in the blood samples, and the concentration of adrenaline in urine were measured. Similar measurements were made before the trips on a normal working day as the control. Phytoncide concentrations in forest and city air were measured. The forest bathing trip significantly increased NK activity and the numbers of NK, perforin, granulysin, and granzyme A/B-expressing cells and significantly decreased the concentration of adrenaline in urine. The increased NK activity lasted for more than 7 days after the trip. In contrast, a city tourist visit did not increase NK activity, numbers of NK cells, nor the expression of selected intracellular anti-cancer proteins, and did not decrease the concentration of adrenaline in urine. Phytoncides, such as alpha-pinene and beta-pinene were detected in forest air, but almost not in city air. These findings indicate that a forest bathing trip increased NK activity, number of NK cells, and levels of intracellular anti-cancer proteins, and that this effect lasted at least 7 days after the trip. Phytoncides released from trees and decreased stress hormone may partially contribute to the increased NK activity.

Forest bathing enhances human natural killer activity and expression of anti-cancer proteins.

In order to explore the effect of forest bathing on human immune function, we investigated natural killer (NK) activity; the number of NK cells, and perforin, granzymes and granulysin-expression in peripheral blood lymphocytes (PBL) during a visit to forest fields. Twelve healthy male subjects, age 37-55 years, were selected with informed consent from three large companies in Tokyo, Japan. The subjects experienced a three-day/two-night trip in three different forest fields. On the first day, subjects walked for two hours in the afternoon in a forest field; and on the second day, they walked for two hours in the morning and afternoon, respectively, in two different forest fields. Blood was sampled on the second and third days, and NK activity; proportions of NK, T cells, granulysin, perforin, and granzymes A/B-expressing cells in PBL were measured. Similar measurements were made before the trip on a normal working day as the control. Almost all of the subjects (11/12) showed higher NK activity after the trip (about 50 percent increased) compared with before. There are significant differences both before and after the trip and between days 1 and 2 in NK activity. The forest bathing trip also significantly increased the numbers of NK, perforin, granulysin, and granzymes A/B-expressing cells. Taken together, these findings indicate that a forest bathing trip can increase NK activity, and that this effect at least partially mediated by increasing the number of NK cells and by the induction of intracellular anti-cancer proteins.

Granulysin-dependent killing of intracellular and extracellular Mycobacterium tuberculosis by Vgamma9/Vdelta2 T lymphocytes.

Contribution of Vgamma9/Vdelta2 T lymphocytes to immune protection against Mycobacterium tuberculosis is still a matter of debate. It was reported earlier that Vgamma9/Vdelta2 T lymphocytes kill macrophages harboring live M. tuberculosis through a granule-dependent mechanism that results in killing of intracellular bacilli. This study found that Vgamma9/Vdelta2 T lymphocytes reduce the viability of both extracellular and intracellular M. tuberculosis. Granulysin and perforin, both detected in Vgamma9/Vdelta2 T lymphocytes, play a major role, which indicates that Vgamma9/Vdelta2 T lymphocytes directly contribute to a protective host response against M. tuberculosis infection.

Immunologic tolerance.

Immunologic tolerance, an active state of antigen specific non-responsiveness, is important in understanding autoimmune diseases and the potential for transplant acceptance. Recent progress in basic studies and preclinical models involving mixed chimerism, costimulatory blockade, immune deviation, and HLA derived peptides suggest that clinical applications are now possible. The National Institutes of Health and Juvenile Diabetes Research Foundation recently launched an Immune Tolerance Network made up of investigators from around the world to develop clinical trials in immune tolerance in autoimmune disease, transplantation and allergy. This review highlights historical perspectives and recent progress in induction and maintenance of immunologic tolerance relevant to pediatric nephrology.

A role of the mitochondrial apoptosis-inducing factor in granulysin-induced apoptosis.

Granulysin is a cytolytic molecule released by CTL via granule-mediated exocytosis. In a previous study we showed that granulysin induced apoptosis using both caspase- and ceramide-dependent and -independent pathways. In the present study we further characterize the biochemical mechanism for granulysin-induced apoptosis of tumor cells. Granulysin-induced death is significantly inhibited by Bcl-2 overexpression and is associated with a rapid (1-5 h) loss of mitochondrial membrane potential, which is not mediated by ceramide generation and is not inhibited by the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Ceramide generation induced by granulysin is a slow event, only observable at longer incubation times (12 h). Apoptosis induced by exogenous natural (C(18)) ceramide is truly associated with mitochondrial membrane potential loss, but contrary to granulysin, this event is inhibited by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Ceramide-induced apoptosis is also completely prevented by Bcl-2 overexpression. The nuclear morphology of cells dying after granulysin treatment in the presence of caspase inhibitors suggested the involvement of mitochondrial apoptosis-inducing factor (AIF) in granulysin-induced cell death. We demonstrate using confocal microscopy that AIF is translocated from mitochondria to the nucleus during granulysin-induced apoptosis. The majority of Bcl-2 transfectants are protected from granulysin-induced cell death, mitochondrial membrane potential loss, and AIF translocation, while a small percentage are not protected. In this small percentage the typical nuclear apoptotic morphology is delayed, being of the AIF type at 5 h time, while at longer times (12 h) the normal apoptotic morphology is predominant. These and previous results support a key role for the mitochondrial pathway of apoptosis, and especially for AIF, during granulysin-induced tumoral cell death.

Granulysin blocks replication of varicella-zoster virus and triggers apoptosis of infected cells.

Granulysin, a lytic protein present in cytolytic granules of human natural killer and cytotoxic T cells, entered cells infected with varicella-zoster virus (VZV). Exposure to granulysin accelerated death of infected cells as assessed by apoptosis markers. The functional domain of granulysin that mediated its antiviral effects was amino acid 23-51; this domain also mediates the additional antitumor cell effects of granulysin. Because granulysin is a product of natural killer cells and T lymphocytes, it is possible that its antiviral activity may act as a mediator of innate and adaptive immune mechanisms.

A distinct pathway of cell-mediated apoptosis initiated by granulysin.

Granulysin is an antimicrobial and tumoricidal molecule expressed in granules of CTL and NK cells. In this study, we show that granulysin damages cell membranes based upon negative charge, disrupts the transmembrane potential (Deltapsi) in mitochondria, and causes release of cytochrome c. Granulysin-induced apoptosis is blocked in cells overexpressing Bcl-2. Despite the release of cytochrome c, procaspase 9 is not processed. Nevertheless, activation of caspase 3 is observed in granulysin-treated cells, suggesting that granulysin activates a novel pathway of CTL- and NK cell-mediated death distinct from granzyme- and death receptor-induced apoptosis.

Granulysin: a novel antimicrobial.

Granulysin is a novel lytic molecule produced by human cytolytic T-lymphocytes (CTLs) and natural killer (NK) cells. It is active against a broad range of microbes, including Gram-positive and -negative bacteria, parasites and Mycobacterium tuberculosis. It is functionally related to other antibacterial peptides, like defensins and magainins, but is structurally distinct. It has structural similarity to porcine NK-lysin and to amoebapores made by Entamoeba histolytica. Synthetic peptides derived from granulysin have differential activity against eukaryotic cells and bacteria. Selective bactericidal peptides may have therapeutic roles as novel antibiotics.

T-cell release of granulysin contributes to host defense in leprosy.

A novel mechanism by which T cells contribute to host defense against microbial pathogens is release of the antimicrobial protein granulysin. We investigated the role of granulysin in human infectious disease using leprosy as a model. Granulysin-expressing T cells were detected in cutaneous leprosy lesions at a six-fold greater frequency in patients with the localized tuberculoid as compared with the disseminated lepromatous form of the disease. In contrast, perforin, a cytolytic molecule that colocalizes with granulysin in cytotoxic granules, was expressed at similar levels across the spectrum of disease. Within leprosy lesions, granulysin colocalized in CD4+ T cells and was expressed in CD4+ T-cell lines derived from skin lesions. These CD4+ T-cell lines lysed targets by the granule exocytosis pathway and reduced the viability of mycobacteria in infected targets. Given the broad antimicrobial spectrum of granulysin, these data provide evidence that T-cell release of granulysin contributes to host defense in human infectious disease.

Granulysin expression is a marker for acute rejection and steroid resistance in human renal transplantation.

Differentiating etiologies of transplant dysfunction without biopsy and optimizing therapy for acute rejection by predicting steroid resistance will reduce patient morbidity. Granulysin is a cytolytic molecule released by CTL and NK cells and coexpressed with effectors of acute allograft rejection, like perforin and granzymes. Granulysin mRNA and protein expression were studied in peripheral blood lymphocytes (PBL; n = 61 total, n = 10 with intercurrent infections) and biopsy tissue from adult and children renal transplant recipients (n = 97) by competitive quantitative-reverse transcriptase-PCR (QC-RT-PCR) and immunohistochemistry. Differences in cell phenotypes were studied in steroid sensitive and resistant acute rejection biopsies. Granulysin was studied in phytohemagglutinin (PHA) stimulated cell lines (donor PBL and CD45RO(+) T cells) by FACS, Western blotting, and RT-PCR after pretreating with cyclosporine A (CSA), azathioprine, mycophenolic acid, and steroids. Granulysin mRNA was significantly increased in patient PBL and transplant biopsies during acute rejection (p < 0.0001) and infection (p < 0.001). Rejecting biopsies alone (n = 53) had mononuclear cell granulysin staining. Steroid resistant biopsies (n = 25) had denser granulysin staining (>2 cells/high power field) and CD45RO(+) lymphocytes, when compared with steroid sensitive (n = 28) rejecting tissue. Granulysin levels were unchanged after azathioprine and mycophenolic acid treatment, decreased after treating activated PBL with steroids and cyclosporine A (CSA), and paradoxically, increased (p < 0.05) after treating CD45RO(+) CTL with CSA. Elevated PBL granulysin is a peripheral marker for acute rejection and infection and dense granulysin staining a tissue marker for steroid resistance. Memory CTL abound in steroid resistant grafts and may have a markedly different response to CSA immunotherapy, suggesting a possible mechanism for steroid resistance.

A non-peptide functional antagonist of the CCR1 chemokine receptor is effective in rat heart transplant rejection.

Chemokines like RANTES appear to play a role in organ transplant rejection. Because RANTES is a potent agonist for the chemokine receptor CCR1, we examined whether the CCR1 receptor antagonist BX471 is efficacious in a rat heterotopic heart transplant rejection model. Treatment of animals with BX471 and a subtherapeutic dose of cyclosporin (2.5 mg/kg), which is by itself ineffective in prolonging transplant rejection, is much more efficacious in prolonging transplantation rejection than animals treated with either cyclosporin or BX471 alone. We have examined the mechanism of action of the CCR1 antagonist in in vitro flow assays over microvascular endothelium and have discovered that the antagonist blocks the firm adhesion of monocytes triggered by RANTES on inflamed endothelium. Together, these data demonstrate a significant role for CCR1 in allograft rejection.

Granulysin, a T cell product, kills bacteria by altering membrane permeability.

Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has antimicrobial activity against a broad spectrum of microbial pathogens. A predicted model generated from the nuclear magnetic resonance structure of a related protein, NK lysin, suggested that granulysin contains a four alpha helical bundle motif, with the alpha helices enriched for positively charged amino acids, including arginine and lysine residues. Denaturation of the polypeptide reduced the alpha helical content from 49 to 18% resulted in complete inhibition of antimicrobial activity. Chemical modification of the arginine, but not the lysine, residues also blocked the antimicrobial activity and interfered with the ability of granulysin to adhere to Escherichia coli and Mycobacterium tuberculosis. Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic ss-galactosidase to its impermeant substrate. By electron microscopy, granulysin triggered fluid accumulation in the periplasm of M. tuberculosis, consistent with osmotic perturbation. These data suggest that the ability of granulysin to kill microbial pathogens is dependent on direct interaction with the microbial cell wall and/or membrane, leading to increased permeability and lysis.

Molecular biology of transplantation.

The past decade has witnessed an explosion in our understanding of the molecules involved in transplant rejection and acceptance. This review highlights our current understanding of antigen recognition, signal transduction, and effector mechanisms of the immune system. Such information has proven important in understanding the action of immunosuppressive drugs and will prove useful in the design of new immunotherapies.

An immunosuppressive and anti-inflammatory HLA class I-derived peptide binds vascular cell adhesion molecule-1.

BACKGROUND: A synthetic peptide corresponding to residues 75-84 of HLA-B2702 modulates immune responses in rodents and humans both in vitro and in vivo. METHODS: We used a yeast two-hybrid screening, an in vitro biochemical method, and an in vivo animal model. RESULTS: Two cellular receptors for this novel immunomodulatory peptide were identified using a yeast two-hybrid screen: immunoglobulin binding protein (BiP), a member of the heat shock protein 70 family, and vascular cell adhesion molecule (VCAM)-1. Identification of BiP as a ligand for this peptide confirms earlier biochemical findings, while the interaction with VCAM-1 suggests an alternative mechanism of action. Binding to the B2702 peptide but not to closely related variants was confirmed by ligand Western blot analysis and correlated with immunomodulatory activity of each peptide. In mice, an ovalbumin-induced allergic pulmonary response was blocked by in vivo administration of either the B2702 peptide or anti-VLA-4 antibody. CONCLUSIONS: We propose that the immunomodulatory effect of the B2702 peptide is caused, in part, by binding to VCAM-1, which then prevents the normal interaction of VCAM-1 with VLA-4.

Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin.

Granulysin, a 9-kDa protein localized to human CTL and NK cell granules, is cytolytic against tumor cells and microbes. Molecular modeling predicts that granulysin is composed of five alpha-helices separated by short loop regions. In this report, synthetic peptides corresponding to the linear granulysin sequence were characterized for lytic activity. Peptides corresponding to the central region of granulysin lyse bacteria, human cells, and synthetic liposomes, while peptides corresponding to the amino or carboxyl regions are not lytic. Peptides corresponding to either helix 2 or helix 3 lyse bacteria, while lysis of human cells and liposomes is dependent on the helix 3 sequence. Peptides in which positively charged arginine residues are substituted with neutral glutamine exhibit reduced lysis of all three targets. While reduction of recombinant 9-kDa granulysin increases lysis of Jurkat cells, reduction of cysteine-containing granulysin peptides decreases lysis of Jurkat cells. In contrast, lysis of bacteria by recombinant granulysin or by cysteine-containing granulysin peptides is unaffected by reducing conditions. Jurkat cells transfected with either CrmA or Bcl-2 are protected from lysis by recombinant granulysin or the peptides. Differential activity of granulysin peptides against tumor cells and bacteria may be exploited to develop specific antibiotics without toxicity for mammalian cells.

Proliferating cell nuclear antigen as the cell cycle sensor for an HLA-derived peptide blocking T cell proliferation.

Synthetic peptides corresponding to structural regions of HLA molecules are novel immunosuppressive agents. A peptide corresponding to residues 65-79 of the alpha-chain of HLA-DQA03011 (DQ65-79) blocks cell cycle progression from early G1 to the G1 restriction point, which inhibits cyclin-dependent kinase-2 activity and phosphorylation of the retinoblastoma protein. A yeast two-hybrid screen identified proliferating cell nuclear Ag (PCNA) as a cellular ligand for this peptide, whose interaction with PCNA was further confirmed by in vitro biochemistry. Electron microscopy demonstrates that the DQ65-79 peptide enters the cell and colocalizes with PCNA in the T cell nucleus in vivo. Binding of the DQ65-79 peptide to PCNA did not block polymerase delta (pol delta)-dependent DNA replication in vitro. These findings support a key role for PCNA as a sensor of cell cycle progression and reveal an unanticipated function for conserved regions of HLA molecules.