Human mast cells produce and release the cytotoxic lymphocyte associated protease granzyme B upon activation.

Mast cells are widely distributed throughout the body and express effector functions in allergic reactions, inflammatory diseases, and host defense. Activation of mast cells results in exocytosis of preformed chemical mediators and leads to novel synthesis and secretion of lipid mediators and cytokines. Here, we show that human mast cells also express and release the cytotoxic lymphocyte-associated protease, granzyme B. Granzyme B was active and localized in cytoplasmic granules, morphologically resembling those present in cytotoxic lymphocytes. Expression and release of granzyme B by mast cell-lines HMC-1 and LAD 2 and by cord blood- and mature skin-derived human mast cells depended on the mode of activation of these cells. In mast cell lines and cord blood-derived mast cells, granzyme B expression was mainly induced by non-physiological stimuli (A23187/PMA, Compound 48/80) and substance P. In contrast, mature skin-derived mast cells only produced granzyme B upon IgE-dependent stimulation. We conclude that granzyme B is expressed and released by human mast cells upon physiologic stimulation. This suggests a role for granzyme B as a novel mediator in mast cell biology.

Activation of the granzyme pathway in children with severe respiratory syncytial virus infection.

Granzymes (Grs), serine proteases present in granules of effector lymphocytes, are involved in several host immune responses, including the activation of cell death and inflammatory pathways. The main goal of this study was to determine whether the local cell-mediated Gr pathway is activated during severe respiratory syncytial virus (RSV) lower respiratory tract illness (LRTI) in children. Tracheal aspirates (TA) from 23 children with RSV-LRTI and 12 controls without pulmonary disease were analyzed for Gr A and B. Bronchoalveolar lavage fluid samples from seven children with RSV-LRTI were analyzed for cellular expression of GrB. Levels of GrA and GrB in TA were significantly increased in RSV patients compared with controls and both Grs showed preserved activity. Gr levels correlated with the total leukocyte counts and IL-8 levels in the airways at several time points. However, no correlation between Gr levels and release of caspase-cleaved cytokeratin-18 was found. There was evidence for marked expression of GrB by both CD8(+) and CD4(+) T cells and natural killer cells in the respiratory tract. These findings suggest activation of the cell-mediated Gr pathway during severe RSV-LRTI in children.

Detection of soluble human granzyme K in vitro and in vivo.

Granzymes are serine proteases released from the granules of cytotoxic lymphocytes during the induction of apoptosis. To evaluate the physiologic role of human granzyme K (GzmK), we developed a sensitive ELISA which was shown to specifically detect human GzmK in its active as well as its inactive conformation. Analysis of the lysate of lymphokine-activated killer (LAK) cells by gel filtration revealed that GzmK seems to be complexed to proteoglycans within these cells. While the expression of GzmA and B by cytotoxic lymphocytes was strongly up-regulated in response to several activating stimuli, GzmK expression did not increase significantly above constitutive levels, indicating differential regulation of these granzymes. However, low levels of GzmK were detected in plasma samples of healthy volunteers, which were in the same range as levels of GzmA and B. Furthermore, circulating levels of GzmK as well as of GzmA and B were significantly elevated in patients suffering from viral infections. We conclude that GzmK protein is produced by cytotoxic cells, and just as GzmA and B it can be released in a soluble form into the extracellular space. Furthermore, our data suggest that despite a more restricted cellular expression pattern, GzmK seems to participate in immune responses against several viruses.

The granzyme B inhibitor proteinase inhibitor 9 (PI9) is expressed by human mast cells.

The activity of granzyme B, a main effector molecule of cytotoxic T lymphocytes (CTL) and natural killer cells, is regulated by the human intracellular serpin proteinase inhibitor 9 (PI9). This inhibitor is particularly expressed by CTL and dendritic cells, in which it serves to protect these cells against endogenous and locally released granzyme B. Moreover, PI9 expression by neoplastic cells may constitute one of the mechanisms for tumors to escape immune surveillance. Here we show that PI9 is also expressed by human mast cells. In immunohistochemical studies using a PI9-specific monoclonal antibody, strong cytoplasmic staining for PI9 was found in normal mast cells in various tissues throughout the body. In addition, in 80% of all cases of cutaneous and systemic mastocytosis tested the majority of the mast cells expressed PI9. As an in vitro model for PI9 expression by mast cells, we studied expression by the human mast cell line HMC-1. Stimulation of HMC-1 with PMA and the calcium ionophore A23187 resulted in a marked increase of PI9 expression. Thus, PI9 is expressed by activated mast cells. We suggest that this expression serves to protect these cells against apoptosis induced by granzyme B released during initiation of the local inflammatory response.

The granzyme B inhibitor SERPINB9 (protease inhibitor 9) circulates in blood and increases on primary cytomegalovirus infection after renal transplantation.

SERPINB9 is the only known human intracellular inhibitor of granzyme B (GrB), the effector molecule in immunity against cytomegalovirus (CMV) and in renal allograft rejection. Therefore, using specific enzyme-linked immunosorbent assays, we addressed the presence of circulating SERPINB9 during primary CMV infection, subclinical rejection, acute rejection, and uncomplicated posttransplantation course. Soluble (s) SERPINB9 circulates in blood and increases on primary CMV infection. This increase was significantly higher in symptomatic than in asymptomatic patients. In contrast, sSERPINB9 levels did not change in response to subclinical or acute rejection. We demonstrated the presence of circulating sSERPINB9/sGrB complexes, which suggests that SERPINB9 has extracellular functions as well.