A CYTOSOLIC GRANZYME-B INHIBITOR RELATED TO THE VIRAL APOPTOTIC REGULATOR CYTOKINE RESPONSE MODIFIER A IS PRESENT IN CYTOTOXIC LYMPHOCYTES

Using a polymerase chain reaction strategy we identified a serine prot einase inhibitor (serpin) in human bone marrow that is related to the cellular serpin proteinase inhibitor 6 (PI-6) and the viral serpin cyt okine response modifier A (CrmA). This serpin, proteinase inhibitor 9 (PI-9), has an unusual reactive center P-1 (Glu)-P-1 '(Cys), which sug gests that it inhibits serine proteinases that cleave after acidic res idues. The only known serine proteinase with this specificity is granz yme B, a granule cytotoxin produced by cytotoxic lymphocytes. To test the interaction of PI-9 with granzyme B we prepared recombinant hexa-h istidine tagged PI-9 in a yeast expression system. Addition of the rec ombinant protein to native granzyme B resulted in an SDS-resistant com plex typical of serpin-serine proteinase interactions. Further analysi s showed that complex formation followed bimolecular kinetics with a s econd order rate constant of 1.7 plus-minus 0.3 times 10(6) M(-1) S-1, which is in the range for a physiologically significant serpin-protei nase interaction. Recombinant PI-9 also completely abrogated granzyme B and perforin-mediated cytotoxicity in vitro. Examination of PI-9 mRN A distribution demonstrated that it it expressed in immune tissue, pri marily in lymphocytes. The highest levels of PI-9 mRNA and protein wer e observed in natural killer cell leukemia cell lines and in interleuk in-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 sepa rate 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 damag ed by their own granzyme B during destruction of abnormal cells.