[Granulysin: antimicrobial molecule of innate and acquired immunity in human tuberculosis]. / Granulysine: médiateur de l'immunité innée et de l'immunité acquise dans la tuberculose chez l'homme.

The recent global increase in cases of tuberculosis and the emergence of multidrug-resistant strains of tuberculosis have focused attention on the molecular mechanisms of human antimycobacterial immunity. The macrophage is not only the primary site for Mycobacterium tuberculosis growth but also ordinarily provides the primary lines of host defense against invading pathogens in its role as an effector of innate immunity. The ability of M. tuberculosis to survive and replicate in the host macrophage is critical to its pathogenesis, emphasizing a need for a clearer understanding of its interactions with the host macrophage. Macrophages use varied strategies to kill and destroy invading organisms, including production of reactive nitrogen and oxygen intermediates, phagosome maturation and acidification, fusion with lysosomes, exposure to defensins and host cell apoptosis. In human, granulysin is a recently identified antimicrobial protein expressed on cytotoxic T cells, natural killer (NK) cells and NKT cells. It has been shown that granulysin contributes to the defense mechanisms against mycobacterial infection. We hypothesized that human macrophages may possess antimicrobial substances, such as granulysin, and play a role in the defense mechanism.

Interferon-gamma-induced membrane PAF-receptor expression confers tumor cell susceptibility to NK perforin-dependent lysis.

Perforin is known to display a membranolytic activity on tumor cells. Nevertheless, perforin release during natural killer (NK)-cell activation is not sufficient to induce membrane target-cell damage. On the basis of the ability of perforin to interact with phospholipids containing a choline phosphate headgroup, we identify the platelet-activating factor (PAF) and its membrane receptor as crucial components in tumor cell killing activity of human resting NK cells. We demonstrate for the first time that upon activation, naive NK cells release the choline phosphate-containing lysolipid PAF, which binds to perforin and acts as an agonist on perforin-induced membrane damage. PAF is known to incorporate cell membranes using a specific receptor. Here we show that interferon-gamma (IFN-gamma) secreted from activated NK cells ends in PAF-receptor expression on perforin-sensitive K562 cells but not on perforin-resistant Daudi cells. In order to prove the capacity of PAF to interact simultaneously with its membrane PAF receptor and with perforin, we successfully co-purified the 3 components in the presence of bridging PAF molecules. The functional activity of this complex was further examined. The aim was to determine whether membrane PAF-receptor expression on tumor cells, driven to express this receptor, could render them sensitive to the perforin lytic pathway. The results confirmed that transfection of the PAF-receptor complementary DNA into major histocompatibility complex class I and Fas-receptor negative tumor cells restored susceptibility to naive NK cells and perforin attack. Failure of IFN-gamma to induce membrane PAF receptor constitutes the first described mechanism for tumor cells to resist the perforin lytic pathway.

Perforin and granzyme B lytic protein expression during chronic viral and autoimmune hepatitis.

BACKGROUND: Cytotoxic T lymphocytes and Kupffer cells are essential components of the immune response during liver diseases. Recent studies have highlighted the role of cytotoxic T lymphocytes using Fas and its ligand in induced hepatocyte death during acute and chronic hepatitis. METHODS: In the present work, the main purpose was to investigate perforin and granzyme B expression in liver biopsies of patients with chronic hepatitis (10 HBV, 14 HCV and 10 autoimmune hepatitis) using immunohistochemistry. The liver biopsies of two normal individuals were also studied in the same conditions. RESULTS: Few intrahepatic T lymphocytes expressed perforin and granzyme B, while a large number of Kupffer cells were positive for both proteins in all the patients tested. The co-localization of perforin and granzyme B, and CD3 or CD68 antigens was visualized, respectively, in T cells and Kupffer cells, using confocal microscopy. In situ hybridization assays confirmed that perforin and granzyme B mRNAs were present in the liver during chronic hepatitis. The results were similar among the three groups of patients and whatever the activity of the disease. Perforin and granzyme B expression was lacking in liver samples from normal individuals. CONCLUSIONS: These data suggest a minor role for the T cell-mediated perforin/granzyme B death pathway, and a putative role for Kuppfer cells via lytic protein release, during chronic hepatitis.

Down-regulation of human granzyme B expression by glucocorticoids. Dexamethasone inhibits binding to the Ikaros and AP-1 regulatory elements of the granzyme B promoter.

The serine protease granzyme B is an essential component of the granule exocytosis pathway, a major apoptotic mechanism used by cytotoxic T lymphocytes and natural killer cells to induce target cell apoptosis. Granzyme B gene transcription is induced in activated lymphocytes upon antigenic stimulation, and several regulatory regions including CBF, AP-1, and Ikaros binding sites have been shown to be essential in the control of granzyme B promoter activation. Dexamethasone, a glucocorticoid that is widely used as an immunomodulatory and anti-inflammatory agent, inhibits granzyme B mRNA transcript in phytohemagglutinin-activated peripheral blood mononuclear cells. Transfection of a reporter construct containing the -148 to +60 region of the human granzyme B promoter demonstrated that this region was the target for dexamethasone repression. Mutation of Ikaros or AP-1 binding sites in the context of the granzyme B promoter demonstrated that both sites participate in dexamethasone-mediated inhibition of the granzyme B promoter activity. Electromobility shift assay revealed that dexamethasone abolished the binding of nuclear transcription factors to the Ikaros binding site and reduced AP-1 binding activity. These results indicate that dexamethasone is able to abrogate the transcriptional activity of the human granzyme B gene promoter by inhibiting the binding of nuclear factors at the AP-1 and Ikaros sites.

[Granzyme B: an essential protease for the inflammatory response]. / Le granzyme B: une protéase essentielle de la réponse inflammatoire.

Granzyme B is a serine protease produced by cytotoxic lymphocytes and capable of inducing rapid target cell death by apoptosis. This effect was found to be closely correlated with the presence of perforin, or pore-forming protein, also contained in the cytoplasmic granules of cytotoxic lymphocytes. Subsequent data suggested that the chief role of perforin is to facilitate accessibility of granzyme B to its cytoplasmic and nuclear targets. Granzyme B may penetrate within the cytoplasm autonomously via a membrane receptor expressed by the target cell, before entering endocytic vesicles containing the protein. Granzyme B can induce target cell death via two complementary pathways, a cytosolic pathway involving cascade activation of proapoptotic caspases, and a nuclear pathway probably involving a cell cycle regulating protein and/or kinase Cdc2 activation. Recent data have established that non-lymphoid cells, including human epithelial cells, can express granzyme B and release it into the extracellular space during the repair process following disruption of the epidermal barrier. This results in local anti-infectious activity that compensates for the break in the mechanical skin barrier and may be a component of natural immunity.

Human peptide transporter deficiency: importance of HLA-B in the presentation of TAP-independent EBV antigens.

Two siblings with a peptide TAP deficiency were recently described. Despite poor cell surface expression of HLA class I molecules, these patients were not unusually susceptible to viral infections. The majority of the cell surface-expressed class I molecules were HLA-B products as assessed by cytofluorometry and biochemical analysis. Analysis of two peptides eluted from the class I molecules expressed by TAP-deficient EBV B lymphoblastoid cell lines indicated that both were derived from cytosolic proteins and presented by HLA-B molecules. Peripheral alphabeta CD8+ T cells were present and their TCR repertoire was polyclonal. Most of the alphabeta CD8+ T cell clones studied (21 of 22) were nonreactive against cells expressing normal levels of the same HLA alleles as those of the TAP-deficient patients. However, it was possible to isolate one cytotoxic CD8+ alphabeta T cell clone recognizing the EBV protein LMP2 presented by HLA-B molecules on TAP-deficient cells. These observations suggest that in the TAP-deficient patients, CD8+ alphabeta T cells could mature and be recruited in immune responses to mediate HLA class I-restricted cytotoxic defense against viral infections. They also strengthen the physiologic importance of a TAP-independent processing pathway of the LMP2 protein, which was previously shown to contain several other TAP-independent epitopes.

CD3- CD56+ non-Hodgkin's lymphomas with an aggressive behavior related to multidrug resistance.

CD56 expression has been reported previously in some non-Hodgkin's lymphoma (NHL) characterization. They principally involve the nasopharynx, are related to Epstein-Barr virus (EBV), and may be classified as either T- or non-T-natural killer (NK) cells according to CD3/T-cell receptor (TCR) status at the genomic or protein level. The present study reports three cases of non-nasal NK-NHL with the following characteristics: an agressive clinical behavior, heterogenous morphological data evoking pleomorphic T-cell malignant lymphoma, a non-T-NK phenotype using flow cytometry, and immunochemistry. The three cases were CD56+ without membrane expression of specific T markers (CD3, CD5, and TCR). Heterogenous results were observed concerning different antigens: CD2, CD4, CD8, CD16, CD94, CD122, TiA1, perforin, and granzyme B. There was no evidence of detectable clonal TCR gene rearrangement with polymerase chain reaction. No NK activity was detected in the two tested cases, and no relation was found with EBV. Multidrug resistance investigations suggest that agressive clinical findings could be related to MDR1 gene expression as confirmed by MDR1 mRNA detection, MDR1 gene product (Pgp) expression, and a functional multidrug resistance study using rhodamine efflux by flow-cytometry.

Acquisition of granzyme B and Fas ligand proteins by human keratinocytes contributes to epidermal cell defense.

In vertebrate tissues, cell integrity is maintained by at least three mechanisms. During an immune response, injured cells are eliminated by cytotoxic lymphoid cells that produce perforin, granzyme B, and Fas ligand (FasL). Second, epithelial cells can produce FasL as an immunosuppressive protein, probably to protect the tissue against immune-mediated damage. Third, locally secreted antimicrobial peptides can be operative in the protection of animal and human epithelia. In this work, as another contribution to local mechanisms of host defense, the ability of human epidermal keratinocytes to produce cytotoxic proteins was investigated. To address this question, freshly isolated human epidermal cells and keratinocytes grown in vitro were studied. Freshly isolated epidermal cells did not express the cytolytic proteins. In contrast, keratinocyte growth to confluence was associated with granzyme B, perforin, and FasL mRNA and protein synthesis. These proteins were secreted in the culture medium. Further analysis showed that they were identical with the ones used by cytotoxic lymphocytes. Their function was then investigated with a view to a potential role in epidermal cell integrity. The data showed that activated human keratinocytes were able to protect against invading pathogens through granzyme B expression. This was demonstrated by the ability of granzyme B to greatly decrease the bacterial growth of Staphylococcus epidermidis. In addition, keratinocytes expressing FasL were found to prevent immune epidermal cell damage. Apoptosis of Fas-sensitive T cells occurred during coculture with confluent epidermal keratinocytes and was largely reduced by the addition of a FasL inhibitor. The data favor keratinocyte involvement in the regulation of dermal inflammatory responses.

Granzyme B and perforin lytic proteins are expressed in CD34+ peripheral blood progenitor cells mobilized by chemotherapy and granulocyte colony-stimulating factor.

Granzyme B and perforin are cytoplasmic granule-associated proteins used by cytotoxic T lymphocytes and natural killer (NK) cells to kill their targets. However, granzyme B gene expression has also been detected in a non-cytotoxic hematopoietic murine multipotent stem cell line, FDCP-Mix. The objective of the present study was to investigate whether granzyme B and perforin could be expressed in human hematopoietic CD34+ cells and if present, discover what their physiologic relevance could be. The primitive CD34+ human cell line KG1a was investigated first and was found to express granzyme B and perforin. Highly purified hematopoietic stem/progenitor cells were then selected using the CD34 surface antigen as marker. Steady-state bone marrow (BM) CD34+ cells did not contain these proteins. Peripheral blood (PB) CD34+ cells, which had been induced to circulate, were also analyzed. After chemotherapy (CT) and granulocyte colony-stimulating factor (G-CSF) treatment, CD34+ cells strongly expressed mRNAs and proteins of granzyme B and perforin. In contrast, CD34+ cells mobilized by G-CSF alone were negative. Western blot analysis further showed that granzyme B and perforin proteins were identical in CD34+ cells and activated PBLs. Such proteins might be implicated in the highly efficient migration of CD34+ stem/progenitor cells from BM to PB after CT and G-CSF treatment. The cellular adhesion mechanisms involved in the BM homing of CD34+ cells are disrupted at least temporarily after CT. The Asp-ase proteolytic activity of granzyme B on extracellular matrix proteins could be used by progenitor cells for their rapid detachment from BM stromal cells and perforin might facilitate their migration across the endothelial cell barrier.

Identification of human granzyme B promoter regulatory elements interacting with activated T-cell-specific proteins: implication of Ikaros and CBF binding sites in promoter activation.

Granzyme B serine protease is found in the granules of activated cytotoxic T cells and in natural and lymphokine-activated killer cells. This protease plays a critical role in the rapid induction of target cell DNA fragmentation. The DNA regulatory elements that are responsible for the specificity of granzyme B gene transcription in activated T-cells reside between nt -148 and +60 (relative to the transcription start point at +1) of the human granzyme B gene promoter. This region contains binding sites for the transcription factors Ikaros, CBF, Ets, and AP-1. Mutational analysis of the human granzyme B promoter reveals that the Ikaros binding site (-143 to -114) and the AP-1/CBF binding site (-103 to -77) are essential for the activation of transcription in phytohemagglutinin-activated peripheral blood lymphocytes, whereas mutation of the Ets binding site does not affect promoter activity in these cells.

Cord blood T lymphocytes lack constitutive perforin expression in contrast to adult peripheral blood T lymphocytes.

Perforin is the cytolytic pore-forming protein, which alone can be responsible for the lethal hit in one of the killing mechanisms used by natural killer (NK) cells or cytotoxic T lymphocytes. In this study, perforin expression was investigated in cord blood (CB) lymphocytes to determine their killing potential in vivo. The majority of CB CD3- NK cells had the protein. Compared with adult perforin-positive NK cells, a significantly lower percentage of cells expressing CD56 and CD57, the related neural cell adhesion molecules, was found (P = .0001). Perforin was also present in a unique immature CB NK-cell subset, characterized by cytoplasmic CD3 antigen (Ag) expression. In CB, very few CD8 perforin-positive T lymphocytes could be detected, but they were in significant numbers in adult peripheral blood (P = .02). A substantial proportion of these cells (70% +/- 23%) lacked the CD28 T-cell coactivation Ag, and they were able to exert NK-like, major histocompatibility complex nonrestricted cytolytic activity. CD4+ and gamma delta-T cells expressing perforin were absent from CB, but low numbers of such cells were detected in adult peripheral blood (P = .0001). Therefore, the spontaneous cytolytic activity of CB lymphocytes appeared to be dependent on well-represented perforin-positive NK cells, which were shown to efficiently lyse NK-sensitive target cells.

Perforin and granzyme B expression is associated with severe acute rejection. Evidence for in situ localization in alveolar lymphocytes of lung-transplanted patients.

To evaluate rejection episodes in lung-transplanted patients, we analyzed 31 bronchoalveolar lavage specimens for lymphocyte levels and lymphocyte expression of two intracytoplasmic activation markers, perforin, the pore-forming lytic protein, and granzyme B, a member of the serine esterase family. Using anti-human granzyme B and perforin mAbs, we show that their expression in alveolar lymphocytes is correlated with the severity of rejection as assessed by histological parameters and the patients' clinical status. The presence of these molecules may provide a prognostic parameter that will facilitate the patients' monitoring, particularly in cases with minimal acute lung rejection susceptible to rapid progression to severe rejection.

Granzyme B and perforin can be used as predictive markers of acute rejection in heart transplantation.

We have investigated perforin and granzyme B expression in graft-infiltrating lymphocytes of patients who underwent heart transplantation. Those proteins are commonly present in the cytoplasmic granules of cytotoxic T lymphocytes and are released upon effector-target cell interaction. From 28 patients 103 endomyocardial biopsies were obtained and examined by histology and immunocytochemical analysis using relevant monoclonal antibodies. We found that "high" biopsy histological grades were associated with perforin and granzyme B expression in graft-infiltrating lymphocytes of patients with acute severe rejection crisis. In contrast, these markers were not detected in patients without rejection or during graft stabilization. Interestingly, in patients with mild rejection and "low" histological grades, two groups could be distinguished with a differential expression of the two intracytoplasmic proteins. The presence of perforin and granzyme B-expressing cells was found to be predictive of rapid progression to severe rejection, so that this situation required additional treatment; in contrast, their absence seemed to correlate with a good graft outcome without additional treatment. Moreover, perforin and granzyme B expression seemed to be down-regulated by immunosuppressive drugs, which coincided with graft stabilization. In conclusion, our data suggest that detection of granzyme B and perforin in graft-infiltrating lymphocytes might be helpful for routinely monitoring heart transplant patients.

A promoter element of the human serine esterase granzyme B gene controls specific transcription in activated T cells.

The human granzyme B gene encodes a serine protease expressed specifically in cytoplasmic granules of cytotoxic T lymphocytes, released upon effector-target cell interaction. Previous studies have shown that granzyme B mRNA was induced in T lymphocytes after antigenic or mitogenic stimulation. To study the regulation of human granzyme B gene expression during lymphocyte activation we analyzed its 5' flanking region using chloramphenicol acetyl transferase (CAT) reporter gene constructs. We show that a 208-bp fragment (-148 to +60) containing an NF-AT (nuclear factor of activated T cells)-binding site promotes CAT expression in phytohemagglutinin-activated T lymphocytes, in immobilized monoclonal anti-CD3 antibody-activated Jurkat T cell line while it is inactive in unstimulated PEER and Jurkat T cells lines or B Epstein-Barr virus-transformed cell lines.

Dissociation of natural killer and lymphocyte-activated killer cell lytic activities in human CD3- large granular lymphocytes.

CD3- large granular lymphocytes (LGL) are known to display natural killer cell (NK) activity without prior sensitization or restriction by major histocompatibility antigens. Upon short-term exposure to interleukin-2, NK cells were shown to acquire lymphocyte-activated killer cell (LAK) activity. The aim of this study was to analyze the characteristics of these lytic activities. Our data indicated that both NK and LAK activities were Ca2+ dependent; however, they could be dissociated by a Ca2+ channel blocker or a Ca2+ channel competitor agent. Moreover, NK activity was associated with granule exocytosis of lytic proteins spontaneously present in CD3- LGL, the most likely candidate being the pore-forming protein perforin. By contrast, LAK activity was found to be dependent on de novo protein synthesis and distinct from granule exocytosis. Our results strongly suggest that NK and LAK activities could be defined as two distinct pathways involving different lytic mediators.

Perforin and granzyme B as markers for acute rejection in heart transplantation.

Histological analysis of endomyocardial biopsies (EMB) is regarded as the most satisfactory technique for monitoring crisis of rejection in heart transplanted patients. In this study, 42 biopsies from 14 patients who underwent heart transplantation were examined. Three patients did not present any rejection crisis at the date of the biopsy analysis, six were examined during an early rejection crisis (day 7-70 post-graft), and five were examined during a late rejection crisis (day 74-960 post-graft). Since granzyme B and perforin are proteins associated with cell lysis histological grading and cell phenotype analysis, in situ hybridization using granzyme B and perforin [35S]RNA probes was performed on 30 EMB to characterize the cytolytic activation of heart infiltrating cells. Our data suggest that granzyme B and perforin could be used as predictive markers for acute rejection in patients with early rejection crisis. Their detection might be an indication to administrate corticoids to resolve an acute rejection crisis. In contrast, their absence in patients with late rejection crisis appears as a good prognostic factor for the outcome of rejection and raises the question of the necessity to treat such patients with additional corticoid treatment.

Structure and evolutionary origin of the human granzyme H gene.

Among the molecules proposed to be involved in cytotoxic T lymphocyte (CTL), natural killer (NK) and lymphokine activated killer (LAK) cell-mediated lysis are the granzymes, a family of serine proteases stored in the cytoplasmic granules of CTLs, NK and LAK cells. In addition to the granzymes A and B, a third member of this family has been cloned in man and designated granzyme H. We present the complete gene sequence including the 5' promoter region and demonstrate that the granzyme H sequence represents a functional gene expressed in activated T cells. Granzyme H shows the highest degree (greater than 54%) of amino acid sequence homology with granzyme B and cathepsin G and, like these genes, consists of five exons separated by introns at equivalent positions. The evolutionary history of granzyme H has been analyzed by reconstructing an evolutionary tree for granzyme sequences. We provide evidence that interlocus recombination between the ancestral genes of granzyme B and granzyme H occurred about 21 million years ago, leading to a replacement of exon 3, intron 3 and part of exon 4 in human granzyme H by human granzyme B sequences. Our results suggest that the ancestral gene of granzyme H is more closely related to cathepsin G and granzyme B than to the murine granzymes C to G. Thus, granzyme H does not represent a human counterpart of the known murine granzymes A to G. It diverged from cathepsin G before mammalian radiation and should, therefore, exist in other mammalian lineages as well.

Perforin and granzyme B: predictive markers for acute GVHD or cardiac rejection after bone marrow or heart transplantation.

Monitoring of human allografts requires to use histological, immunohistochemical and functional techniques to characterize graft infiltrating cells. Granzyme B and perforin gene expression is of major importance in functional studies. Those proteins are present in the cytoplasmic granules of cytotoxic T lymphocytes and are secreted during granule exocytosis at the effector/target cell interface. Gene expression of both proteins has been studied by in situ hybridization using specific riboprobes on serial sections of biopsies in two pathological models. Our results show that cells infiltrating early skin lesions of patients with acute GVHD after bone marrow graft are exclusively composed of T cells, among which some of them express granzyme B and perforin genes. Similarly the presence of granzyme B and perforine-expressing cells in endomyocardial biopsies of heart transplanted patients has been associated to early and severe crisis of rejection. In contrast, the absence of functional markers in lymphoid infiltrates was coinciding with less aggressive and late episodes of rejection. Taken together, our data indicate that granzyme B and perforin gene expression in skin infiltrating lymphocytes during GVH or within heart infiltrating cells during crisis of rejection are in favor of severe processes. The study has allowed to predict during heart transplantation the apparition of a rejection crisis and to show the necessity for treating the patient with immunsuppresive drugs. This is also the case for patients with GVHD at the time of the first skin rash.

Involvement of granzyme B and perforin gene expression in the lytic potential of human natural killer cells.

Natural killer (NK) cells (CD3-) or large granular lymphocytes (LGL) spontaneously kill K562 targets but are unable to kill Daudi cells in the absence of IL-2 stimulation. IL-4 is reported to prevent or inhibit the IL-2-driven lymphokine-activated killer (LAK) generation in NK cells. Therefore, we wished to determine whether the antagonistic effect of IL-4 on IL-2-induced LAK activity might regulate the expression of genes encoding proteins involved in lysis, such as perforin, the pore-forming protein, or which are associated with lysis, such as granzymes A and B. By using in situ hybridization, we showed that, in addition to inducing LAK activity, IL-2 stimulation increased the amount of perforin and granzyme B mRNA at the single-cell level in 40 to 100% of the total CD3- LGL cell population. In addition, our results indicated that the stimulatory effect of IL-2 can be downregulated by IL-4 for both LAK activity and granzyme B and perforin gene expression. Here again, a decrease in the amount of specific mRNA per cell was noted. These findings suggest that modulation of the lytic machinery via lymphokines might be associated with regulation of the lytic potential of NK cells.