Granzyme activity in the inflamed lung is not controlled by endogenous serine proteinase inhibitors.

Numerous lung diseases, such as hypersensitivity pneumonitis (HP), are characterized by the presence of activated alveolar CTL and NK cells. Since these cells produce granzymes, granzyme A and B levels in bronchoalveolar lavage (BAL) fluids from 14 normal subjects and 12 patients with HP were measured by ELISA. Median (range) BAL granzyme A and B levels were 4 (0-37) and 0 (0-6) pg/ml in normal subjects. BAL granzyme levels were significantly higher in HP patients, being at 74 (0-1,889) and 10 (0-78) pg/ml for granzymes A and B, respectively. In vitro, neither of the three main serine protease inhibitors of the lung, namely alpha1-antitrypsin, secretory leukocyte protease inhibitor, and elafin, showed any effect on granzyme A or B activity. In addition, granzyme A was shown to be fully active in BAL fluids. Hence, these data show that granzyme activity may be poorly controlled by protease inhibitors in inflamed tissues. Thus, granzymes could contribute to tissue remodeling and inflammation characterizing HP.

Soluble granzymes are released during human endotoxemia and in patients with severe infection due to gram-negative bacteria.

Extracellular release of granzymes is considered to reflect the involvement of cytotoxic T lymphocytes and NK cells in various disease states. To obtain insight into granzyme release during bacterial infection, granzyme levels were measured during experimental human endotoxemia and in patients with melioidosis, a severe infection due to gram-negative bacteria. Plasma concentrations of granzyme A (GrA) and GrB increased transiently after endotoxin administration, peaking after 2-6 h. In patients with bacteremic melioidosis, GrA and GrB levels were elevated on admission and remained high during the 72-h study period. In whole blood stimulated with heat-killed Burkholderia pseudomallei, neutralization of tumor necrosis factor, interleukin-12, or interleukin-18 inhibited granzyme secretion, which was independent of interferon-gamma. Stimulation with endotoxin and other gram-negative and gram-positive bacteria also strongly induced the secretion of granzymes, suggesting that granzyme release is a general immune response during bacterial infection. The interaction between the cytokine network and granzymes may play an important immunoregulatory role during bacterial infections.

Expression of granzyme B during primary cytomegalovirus infection after renal transplantation.

CD8+ T cells employ granzyme B (GrB) to induce apoptosis in target cells. Increased expression of GrB has been put forward as a diagnostic marker in transplant rejection and viral infection. Three-color flow cytometric analysis revealed that peripheral blood CD8+ T lymphocytosis during primary cytomegalovirus infection after renal transplantation resulted from expansion of a CD8+GrB+CD62L+ T cell subset that was almost absent during stable transplant function or acute rejection. This expansion coincided with a temporary increase in systemic soluble GrB (sGrB) levels. No such increase was observed during stable transplant function or acute rejection. Thus, the primary immune response to cytomegalovirus infection is accompanied by appearance of CD8+GrB+CD62L+ T cells and increased sGrB levels in the peripheral blood compartment. Determination of the latter may provide a novel approach for monitoring viral infections.

The CD8+ granzyme B+ T-cell subset in peripheral blood from healthy individuals contains activated and apoptosis-prone cells.

Granzyme B (GrB) has been implicated in induction of apoptosis in target cells. The presence of GrB in peripheral blood CD8+ T cells from healthy individuals was analysed in immunocytochemical and flow cytometric studies. Furthermore, CD8+ GrB- T cells and CD8+ GrB+ T cells were compared regarding phenotypical characteristics and susceptibility to both spontaneous and Fasmediated apoptosis. GrB was expressed by approximately one-fifth of CD8+ T cells. Compared with the CD8+ GrB- T-cell subset, the CD8+ GrB+ T-cell subset contained cells that were relatively more activated and more prone to spontaneous apoptosis. Culturing of cells with immunoglobulin M (IgM) anti-Fas monoclonal antibody had no additional effect on the number of CD8+ GrB+ T cells undergoing apoptosis. We suggest that the presence of CD8+ GrB+ T cells in peripheral blood from healthy individuals results from immune surveillance or contact with infectious agents, and that spontaneous apoptosis of these cells might serve as a mechanism for their eventual clearance.

Extracellular granzymes A and B in humans: detection of native species during CTL responses in vitro and in vivo.

Activated CTLs and NK cells induce apoptosis via multiple mechanisms, including that termed granule exocytosis. The latter pathway consists of vectorial secretion of perforin and a family of granule-associated serine proteases (granzymes) to the target cell. To establish whether granzymes are released extracellularly during cytolytic reactions in vivo, ELISAs that measure the native enzymes were developed and were found to specifically detect granzyme A (GrA) and granzyme B (GrB) at picogram concentrations. Low levels of GrA and GrB were present in plasma of healthy individuals (GrA, 33.5 pg/ml (median); GrB, 11.5 pg/ml (median)), whereas significantly higher levels were present in patients with ongoing CTL response, i.e., patients suffering from infections by EBV or HIV type 1. Markedly elevated levels were also noted in synovial fluid of patients with active rheumatoid arthritis. The measurement of soluble granzymes should be useful to assess clinical disorders associated with activated CTL and NK cells. Furthermore, these results suggest that granzymes mediate biologic effects beyond their described role in apoptotic cell death.