A secreted preform of neutrophil proteinase 3 regulates the proliferation of granulopoietic progenitor cells

Myeloid leukemia cells, the human promyelocytic cell line HL-60, and a subp opulation of normal marrow cells produce a leukemia-associated inhibitor (L AI) that reversibly downmodulates DNA synthesis of normal granulopoietic pr ogenitor cells colony-forming unit granulocyte-macrophage (CFU-GM). We isol ated an active 125-kD component of LAI from HL-60 conditioned medium (CM), subjected it to cyanogen bromide cleavage and show by amino acid sequencing of the resulting peptides that it consists of a complex of the serine prot einase inhibitor alpha 1-antitrypsin and a 31-kD fragment that retained the S-phase inhibitory activity, but resisted sequencing. This finding suggest ed that the 31-kD fragment originated from one of the neutrophil serine pro teases (ie, elastase, proteinase 3, or cathepsin G) produced by normal prom yelocytes, as well as HL-60 cells, for storage in primary granules and part ly secreted during synthesis as enzymatically inactive proforms. Immunoblot analysis showed that the 125-kD complex contained proteinase 3 (PR3), and immunoprecipitation of PR3 from HL-60 CM abrogated the S-phase inhibitory a ctivity, whereas immunoprecipitation of cathepsin G or elastase did not. Im munoprecipitation of PR3 from CM of a subpopulation of normal marrow cells also abrogated the S-phase inhibitory effect. Furthermore, CM from rat RBL and murine 32D cell lines transfected with human PR3 both reduced the fract ion of CFU-GM in S-phase with 30% to 80% at 1 to 35 ng/mL PR3, whereas CM o f the same cells transfected with cathepsin G or elastase did not. Also, an enzymatically silent mutant of PR3 exerted full activity, showing that the S-phase modulatory effect is not dependent on proteolytic activity. Amino acid sequencing of biosynthetically radiolabeled PR3 showed that PR3 from t ransfected cells is secreted after synthesis as proforms retaining amino te rminal propeptides. In contrast, mature PR3 extracted from mature neutrophi ls has only minor activity. The inhibitory effect of secreted PR3 is revers ible and abrogated by granulocyte (G)- or granulocyte-macrophage colony-sti mulating factor (GM-CSF). Experiments with highly purified CD34(+) bone mar row cells suggested that PR3 acts directly on the granulopoietic progenitor cells. These observations suggest a role for PR3 in regulation of granulop oiesis, and possibly in suppression of normal granulopoiesis in leukemia. ( C) 1999 by The American Society of Hematology.