Immunology: Expression of perforin, granzyme A and TIA-1 by human uterine CD56+ NK cells implies they are activated and capable of effector functions

At the time the human placenta is established, the uterine mucosallining (decidua) is infiltrated by abundant CD3^– CD56^brightnatural killer (NK) cells. NK cells circulating in blood areknown to contain perforin and granzyme A in their cytoplasmicgranules. TIA-1, an RNA-binding protein capable of inducingDNA fragmentation, has also been found in the granules of cytolyticcells. In this paper, we demonstrate the presence of perforin,granzyme A and TIA-1 in the granules of uterine NK cells. Sixteensections of non-pregnant endometrium throughout the menstualcycle and six sections of early decidua, together with cytospinsof four preparations of isolated decidual leukocytes were stainedby both immuno-histology and immuno-electron microscopy to localizeperforin, granzyme A and TIA-1 to the cytoplasmic granules ofCD56⁺ cells. The presence in vivo of these cytolytic moleculesin a normal physiological situation implies that these uterineNK cells may have effector functions in the control of normalplacentation.     

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.     

Serial killing by cytotoxic T lymphocytes: T cell receptor triggers degranulation,re-filling of the lytic granules and secretion of lytic proteins via a non-granule pathway

CD8⁺ cytotoxic T lymphocyte (CTL) clones begin to synthesize the lytic proteins granzyme A, granzyme B and perforin after stimulation with allogeneic target cells. The lytic proteins are stored in the secretory granules which are released after cross-linking of the T cell receptor (TcR) upon target cell recognition. During lytic granule biogenesis granzyme A protein synthesis can be detected between 2 and 10 days after allogeneic stimulation of the CTL. Although granzyme A is stored in the lytic granules over this period, the majority of granzyme A synthesized is secreted directly from the CTL. TcR triggering of degranulation also results in new synthesis of the lytic proteins, which can be inhibited by cycloheximide (CHX). Some of the newly synthesized lytic proteins can be stored in the cell and refill the granules. But up to one third of granzymes A and B can be secreted directly from the CTL via the constitutive secretory pathway as shown by granzyme A enzymatic activity and immunoblots of secreted granzyme B, where one third of the protein fails to acquire the granule targeting signal. Perforin is also secreted via the constitutive pathway, both from the natural killer cell line, YT, and from CTL clones after TcR cross-linking. Constitutive secretion of the lytic proteins can be blocked by both CHX and brefeldin A (BFA). While BFA does not affect the directional killing of recognized targets, it abrogates bystander killing, indicating that bystander killing arises from newly synthesized lytic proteins delivered via a non-granule route. These results demonstrate that the perforin/granzyme-mediated lytic pathway can be maintained while CTL kill multiple targets. We show that CTL not only re-fill their granules during killing, but also secrete lytic proteins via a non-granule-mediated pathway.     

Expression of granzyme B in peripheral blood polymorphonuclear neutrophils (PMN), myeloid cell lines and in PMN derived from haemotopoietic stem cells in vitro

Granzyme B and perforin are the major protagonists of cytotoxicity mediated by natural killer (NK) cells or cytotoxic T cells. More recent we described the presence of granzyme B and perforin in polymorphonuclear neutrophils (PMN), a finding in discrepancy with the credo that granzyme B and perforin expression is restricted to cytotoxic T cells and NK cells. In extension of our previous study, we now provide evidence that granzyme B is not only present in mature PMN, but also in the myeloid cell lines HL-60 and U937, in CD34+ stem cells, and in PMN derived from CD34+ cells in vitro. In agreement with the "targeting by time" hypothesis we found the bulk of granzyme B in association with primary granules, in addition to a minor membrane expression. Granzyme B, on one hand might, enhance the cytotoxic potential of PMN, on the other, it may provide PMN with additional means to degrade extracellular matrices.     

Human granzyme B is essential for DNA fragmentation of susceptible target cells

We have partially characterized the granules of the human NK cell line, YT-INDY, and assessed granule-mediated lysis and DNA fragmentation of assorted targets. Biochemical studies demonstrated significant quantities of granzyme B (asp-ase) and a heretofore undescribed chymase but no tryptase (i.e., granzyme A or 3) or distinct met-ase. YT-INDY expressed mRNA for granzyme B, perforin and CCPX. The existence of perforin was confirmed by immunoblot. The granules lysed both human and murine NK-sensitive and NK-resistant targets. YTindependent lytic pathway is associated with the granules. In addition, 4-(2-aminoethyl) benzenesulfonylfluoride hydrochloride (AEBSF), an inhibitor that selectively blocked the chymase and 3,4-dichloroisocoumarin (DCI), an inhibitor that inactivated both chymase and asp-ase activities, marginally affected lysis. By gel electrophoresis and ¹²⁵I-labeled deoxyuridine release assay, only murine cells (SP2/0 and YAC-1) underwent DNA fragmentation, and cleavage was completely inhibited by DCI, whereas EGTA, AEBSF and aurintricarboxylic acid (ATA) had no effect. The results, therefore, underscore the central role of granzyme B in granule-mediated DNA fragmentation, emphasize that the protease acts via an ATA-resistant endonuclease pathway and stress that nucleolysis does not invariably accompany granule-mediated cytolysis. Finally, ATA inhibited the asp-ase activity of isolated but not granule-associated granzyme B. ATA, therefore, is not a specific endonuclease inhibitor and results obtained with ATA should be viewed cautiously.     

CD56bright cells differ in their KIR repertoire and cytotoxic features from CD56dim NK cells

In this study we present new differential characteristics of NK cells expressing CD56 surface antigen in low (CD56dim) or high (CD56bright) density. In contrast to CD56bright NK cells CD56dim cells express killer cell immunoglobulin (Ig)-like receptors (KIR) such as CD158a, CD158b, and NKB1. However, c-type lectin-like receptors (KLR) CD94/NKG2 and CD161 are present on both subsets. The ability to form conjugates with susceptible targets is approximately twice as strongly pronounced in CD56dim vs. CD56bright NK cells. Last but not least, granules of CD56dim cells contain about tenfold more perforin and granzyme A enabling potentially more effective cytolysis compared to CD56bright NK cells. On the other hand, CD56bright NK cells are superior in producing the proinflammatory cytokines IFN-gamma (28.5% vs. 20.8%, p<0.05) and TNF-alpha (28% vs. 15.8%, p<0.001). The different NK cell populations retained their specific phenotype in vitro during culture in the presence of IL-2 contradicting that they simply display different stages of maturity. Taken together our data support the view that CD56bright cells are specialized NK cells that regulate immunological response mechanisms rather by cytokine supply than by their cytotoxic potential. The poor cytolytic capacity of CD56bright NK cells can be explained by weak ability in forming conjugates with target cells and low contents of perforin and granzyme A in their granules.     

Human yeast-specific CD8 T lymphocytes show a nonclassical effector molecule profile

Pathogenic yeast and fungi represent a major group of human pathogens. The consequences of infections are diverse and range from local, clinically uncomplicated mycosis of the skin to systemic, life-threatening sepsis. Despite extensive MHC class I-restricted frequencies of yeast-specific CD8 T lymphocytes in healthy individuals and the essential role of the cell-mediated immunity in controlling infections, the characteristics and defense mechanisms of antifungal effector cells are still unclear. Here, we describe the direct analysis of yeast-specific CD8 T lymphocytes in whole blood from healthy individuals. They show a unique, nonclassical phenotype expressing granulysin and granzyme K in lytic granules instead of the major effector molecules perforin and granzyme B. After stimulation in whole blood, yeast-specific CD8 T cells degranulated and, upon cultivation in the presence of IL-2, their granula were refilled with granulysin rather than with perforin and granzyme B. Moreover, yeast-specific stimulation through dendritic cells but not by yeast cells alone led to degranulation of the effector cells. As granulysin is the only effector molecule in lytic granules known to have antifungal properties, our data suggest yeast-specific CD8 T cells to be a nonclassical effector population whose antimicrobial effector machinery seems to be tailor-made for the efficient elimination of fungi as pathogens.     

Analysis of Perforin Expression in Human Peripheral Blood Lymphocytes,CD 56+ Natural Killer Cell Subsets and Its Induction by Interleukin-2

In this study new evidence is obtained by the use of an anti human perforin monoclonal antibody (mAb), anti P1, concerning the number of perforin positive cells in human peripheral blood lymphocytes (PBL). It is shown that about 23% of PBL is perforin postive and that this percent increases by the treatment in RPMI 1640 medium alone to 33% and with 1000 U r hIL-2 to 46%. Assessment of the cytotoxicity potential of NK cells from PBL, freshly isolated and treated, against tumor cell line K562 by the standard NK cell 4-hr 51-chromium release assay, indicates a significant enhancement in their cytotoxicity. By FACStar sorting and analysis of the CD56+ NK cell population new evidence is obtained which shows that about 25–30% of this population represents the CD56^bright+ subset, while 70–75% represents the CD56^dim+ subset. As the two subsets were shown to differ functionally they were stained with anti P1 for the evaluation of perforin content and it was found that both of them are positive for perforin from 97–99%, suggesting that the functional difference is not due to perforin content. In this sense, as NK cells are constitutively positive for perforin, the increase in the cytotoxicity of NK cells induced by IL-2 is most likely due to the synthesis and expression of various adhesion molecules on NK cells which increase their cytotoxic potential, as well as, that the detected increase in the number of perforin postive cells by this lymphokine does not belong to NK, but to the T lymphocyte population. The data obtained in this study indicate the possibility of perforin detection in human lymphocytes by an anti human perforin mAb and the change in the number of perforin positive cells after stimulation with interleukin-2.     

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.     

Impaired circulating CD56dim NK cells are associated with decompensation of HBV-related cirrhosis

NK cells play an important role in immune regulation and defense of infection, but their characteristics in patients with decompensated cirrhosis and their relationship with liver function remain unclear. We studied the functional properties of NK cells (including CD56^dim NK and CD56^bright NK cells) in patients with HBV-related decompensated liver cirrhosis (HBV-DLC) and analyzed their relationship with decompensation of liver function. Thirty patients with HBV-DLC and 25 patients with HBV-related compensated liver cirrhosis (HBV-CLC) were recruited in this study. Twenty five age- and sex-matched healthy individuals were recruited as healthy controls (HCs). The phenotypical and functional characteristics of NK cell subsets were detected by flow cytometry, and the correlation between NK cells and decompensation of liver function was analyzed. The frequency of circulating CD56^bright NK cells was significantly increased while circulating CD56^dim NK cells was significantly decreased in HBV-DLC patients as compared with HCs and HBV-CLC patients. Peripheral activated-CD56^bright NK cells from HBV-DLC patients might express lower levels of inhibitory receptor CD158b1/2 and higher levels of activating receptor NKG2D and their expression of perforin and granzyme A/B also increased significantly compared with HCs, suggesting a high immune activation status of peripheral CD56^bright NK cells in HBV-DLC patients. In HBV-DLC patients, the expression of CD107a and perforin in circulating CD56^dim NK cells was positively correlated with cytolytic capacity while CD107a and perforin expression in circulating CD56^dim NK cells were significantly decreased, suggesting an impaired cytolytic capacity of circulating CD56^dim NK cells. Besides, we found that the perforin expression of circulating CD56^dim NK cells correlated negatively with child-pugh classification in HBV-DLC patients. The functional properties of circulating NK cell subsets in HBV-DLC patients have changed significantly, especially of CD56^dim NK cells which closely related to decompensation of liver function. These findings may help provide new perspectives and theoretical basis for the treatment of patients with HBV-DLC.     

Absence of cytotoxic molecules in CD8- and/or CD56-positive adult T-cell leukaemia/lymphoma

Adult T-cell leukaemia/lymphoma (ATLL) cells usually exhibit a CD4⁺ (helper/inducer) phenotype (CD4⁺/8^–/56^–), and only a minority of tumours express the CD8 (cytotoxic/suppressor) or CD56 (natural killer [NK]-associated) antigens. TIA-1 is a cytotoxic granule-associated protein expressed in NK cells and cytotoxic T lymphocytes (CTLs). Granzyme B, perforin and Fas ligand (FasL) are also expressed in activated CTLs and NK cells. To clarify the cytotoxic potential of ATLL cells, immunohistochemistry was performed in CD8⁺ and/or CD56⁺ ATLL cells, using anti-TIA-1, anti-granzyme B, anti-perforin and anti-FasL antibodies. We studied nine cases of CD8⁺ and/or CD56+ ATLL, all of which exhibited monoclonal integration of human T-cell leukaemia virus type 1 (HTLV-1) proviral DNA. Four cases exhibited a CD8⁺/CD56^– phenotype, four others had a CD8^–/CD56⁺ phenotype, and one was CD8⁺/CD56⁺. All but one case also expressed the surface antigens CD3, TCR αβ, and CD4. Expression of granzyme B and TIA-1 were demonstrated in three and two cases, respectively, but none expressed perforin or FasL. In the control study, 10 cases with typical CD3⁺/4⁺/8^–/56^– ATLL demonstrated no expression of those cytotoxic-associated proteins. Our findings suggest that CD8 and/or CD56 positivity probably confer(s) no cytotoxic function on ATLL cells, and it is possible that CD8 and CD56 may be simply aberrant surface markers in ATLL. Received: 20 January 1999 / Accepted: 13 April 1999     

Differential Expression of Perforin in Cytotoxic Lymphocyte in HIV/AIDS Patients of China

Cytotoxic lymphocytes are critical in the control of HIV replication, it has been shown that perforin is the key effector of killing machinery for CTLs and NK cells, so we investigated the circulating levels of perforin in CD8⁺T cells and NK cells by flow cytometry intracellular stain in Chinese HIV infected individuals, its association with disease progression was analyzed. Our results showed that NK cells express perforin more efficiently than CD8⁺T cells, CD8⁺T cells expressed perforin higher than that of healthy controls, but NK cells expressed lower perforin than that of healthy controls, both were not correlated with disease progression. but significantly associated with their numbers, anti-retrovirus therapy had no evident effects on peforin expression in CD8⁺T cells, but enhanced perfrin expression in NK cells, perforin expression in CD8⁺T cells and CD16⁺NK cells correlate with CD4⁺T cell counts significantly in HAART-treated group. Therefore, different mechanisms may be involved in regulating peripheral perforin expression in different cell types.     

Phytoncides (wood essential oils) induce human natural killer cell activity

To explore the effect of forest bathing on the human immune system, we investigated the effect of phytoncides (wood essential oils) on natural killer (NK) activity and the expression of perforin, granzyme A and granulysin in human NK cells. We used NK-92MI cell, an interleukin-2 independent human NK cell line derived from the NK-92 cell, in the present study. NK-92MI cells express the CD56 surface marker, perforin, granzyme A, and granulysin by flow cytometry and are highly cytotoxic to K562 cells in chromium release assay. Phytoncides significantly increase cytolytic activity of NK-92MI cells in a dose-dependent manner and significantly increase the expression of perforin, granzyme A, and granulysin in the NK-92MI cells. Phytoncides also partially, but significantly, restore the decreased human NK activity and the decreased perforin, granzyme A, and granulysin expression in NK-92MI cells induced by dimethyl 2,2-dichlorovinyl phosphate (DDVP), an organophosphorus pesticide. Pretreatment with phytoncides partially prevents DDVP-induced inhibition of NK activity. Taken together, these data indicate that phytoncides significantly enhance human NK activity and this effect is at least partially mediated by induction of intracellular perforin, granzyme A, and granulysin.     

Phytoncides (Wood Essential Oils) Induce Human Natural Killer Cell Activity

To explore the effect of forest bathing on the human immune system, we investigated the effect of phytoncides (wood essential oils) on natural killer (NK) activity and the expression of perforin, granzyme A and granulysin in human NK cells. We used NK-92MI cell, an interleukin-2 independent human NK cell line derived from the NK-92 cell, in the present study. NK-92MI cells express the CD56 surface marker, perforin, granzyme A, and granulysin by flow cytometry and are highly cytotoxic to K562 cells in chromium release assay. Phytoncides significantly increase cytolytic activity of NK-92MI cells in a dose-dependent manner and significantly increase the expression of perforin, granzyme A, and granulysin in the NK-92MI cells. Phytoncides also partially, but significantly, restore the decreased human NK activity and the decreased perforin, granzyme A, and granulysin expression in NK-92MI cells induced by dimethyl 2,2-dichlorovinyl phosphate (DDVP), an organophosphorus pesticide. Pretreatment with phytoncides partially prevents DDVP-induced inhibition of NK activity. Taken together, these data indicate that phytoncides significantly enhance human NK activity and this effect is at least partially mediated by induction of intracellular perforin, granzyme A, and granulysin.     

Cytolytic effector function is present in resting peripheral T lymphocytes.

Antigen-specific cytotoxic killer lymphocytes (CTLs) represent one of the major effector functions of the immune system. It is well established that, as a consequence of TCR recognition of the antigen-bearing target cell, resting T lymphocytes develop into fully active antigen-specific CTLs. In contrast, natural killer (NK) cells are immediately lytic upon contact with an appropriate target cell. The lytic machinery of CTLs and NK cells is thought to include the contents of their cytoplasmic granules, in particular the pore-forming protein perforin. Here we report direct cytolytic activity by resting peripheral CD3+CD8+ T cells as a result of TCR-CD3 binding to the target cell; the murine OKT3 hybridoma (anti-human CD3) was used as a target. The cytotoxicity was more pronounced in the CD8+CD45RO+ population, which contains 'memory' T cells, than in the reciprocal CD8+CD45RA+ subset; CD8+CD4- mature thymocytes were non-cytotoxic. The cytolytic potential of these populations correlated with the presence or absence of perforin. The results demonstrate that the cytolytic machinery of T cells develops post-thymically and can be immediately triggered by TCR-CD3 stimulation.     

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.