A Cytosolic Granzyme B Inhibitor Related to the Viral Apoptotic Regulator Cytokine Response Modifier A Is Present in Cytotoxic Lymphocytes

Abstract

Using a polymerase chain reaction strategy we identified a serine proteinase inhibitor (serpin) in human bone marrow that is related to the cellular serpin proteinase inhibitor 6 (PI-6) and the viral serpin cytokine response modifier A (CrmA). This serpin, proteinase inhibitor 9 (PI-9), has an unusual reactive center P 1 (Glu)-P 1 (Cys), which suggests that it inhibits serine proteinases that cleave after acidic residues. The only known serine proteinase with this specificity is granzyme B, a granule cytotoxin produced by cytotoxic lymphocytes. To test the interaction of PI-9 with granzyme B we prepared recombinant hexa-histidine tagged PI-9 in a yeast expression system. Addition of the recombinant protein to native granzyme B resulted in an SDS-resistant complex typical of serpin-serine proteinase interactions. Further analysis showed that complex formation followed bimolecular kinetics with a second order rate constant of 1.7 ؎ 0.3 ؋ 10 6 M ؊1 s ؊1, which is in the range for a physiologically significant serpin-proteinase interaction. Recombinant PI-9 also completely abrogated granzyme B and perforin-mediated cytotoxicity in vitro. Examination of PI-9 mRNA distribution demonstrated that it is expressed in immune tissue, primarily in lymphocytes. The highest levels of PI-9 mRNA and protein were observed in natural killer cell leukemia cell lines and in interleukin-2 stimulated peripheral blood mononuclear cells, which also produce granzyme B. Like PI-6, PI-9 was shown to be a cytosolic protein that is not secreted. Fractionation of natural killer cells and stimulated peripheral blood mononuclear cells demonstrated that PI-9 is in a separate subcellular compartment to granzyme B. These results suggest that PI-9 serves to inactivate misdirected granzyme B following cytotoxic cell degranulation. This may explain why cytotoxic cells are not damaged by their own granzyme B during destruction of abnormal cells.

The Granzyme B Inhibitor, Protease Inhibitor 9, Is Mainly Expressed by Dendritic Cells and at Immune-Privileged Sites

“…It was previously reported that in addition to CTLs PI9 is particularly produced in B-lymphocytes (12). These conclusions were based on analysis of PI9 protein expression in EBV-transformed B cell lines.…”

“…Recently, a novel human intracellular serpin, proteinase inhibitor (PI) 9, was described to be expressed by CTLs. This serpin efficiently inhibits granzyme B in vitro and in vivo and, hence, PI9 transfected cells are protected against granzyme B mediated apoptosis (11,12). Using RT-PCR and Northern blotting it was shown that PI9 mRNA is mainly expressed by B and T cell lines and, therefore, it was proposed that PI9 protects CTLs against death induced by their own misdirected granzyme B (11).…”

Serpin 2a Is Induced in Activated Macrophages and Conjugates to a Ubiquitin Homolog

“…Presumably, the cytoplasmic PI-6 protects the neutrophil from granule rupture before release from the cell. PI-9, described above, is expressed by cytotoxic T cells and NK cells, which also express the PI-9 target, granzyme B, in their cytotoxic granules (36). It has been proposed by Bird et al (38) that PI-9 protects T cells and NK cells against misdirected granzyme B after degranulation or leakage of granzyme B from cytotoxic granules within the cell.…”

Expression of Cytotoxic Proteins by Neoplastic T Cells in Mycosis Fungoides Increases with Progression from Plaque Stage to Tumor Stage Disease

“…32 Other studies demonstrated that CTL can escape from GrB-mediated apoptosis by expressing intracellular serine protease inhibitors (serpins). 33 One could speculate that similar mechanisms are operative not only in CTL and NK cells but also in tumor cells expressing these cytotoxic proteins. This would imply that such tumor cells might be rather resistant against perforin/granzyme B-mediated, eg.…”