Neutrophil granulocytes form the body’s first line of antibacterial defense, but they also contribute to tissue injury and noninfectious, chronic inflammation. Proteinase 3 (PR3) and neutrophil elastase (NE) are 2 abundant neutrophil serine proteases implicated in antimicrobial defense with overlapping and potentially redundant substrate specificity. Here, we unraveled a cooperative role for PR3 and NE in neutrophil activation and noninfectious inflammation in vivo, which we believe to be novel. Mice lacking both PR3 and NE demonstrated strongly diminished immune complex–mediated (IC-mediated) neutrophil infiltration in vivo as well as reduced activation of isolated neutrophils by ICs in vitro. In contrast, in mice lacking just NE, neutrophil recruitment to ICs was only marginally impaired. The defects in mice lacking both PR3 and NE were directly linked to the accumulation of antiinflammatory progranulin (PGRN). Both PR3 and NE cleaved PGRN in vitro and during neutrophil activation and inflammation in vivo. Local administration of recombinant PGRN potently inhibited neutrophilic inflammation in vivo, demonstrating that PGRN represents a crucial inflammation-suppressing mediator. We conclude that PR3 and NE enhance neutrophil-dependent inflammation by eliminating the local antiinflammatory activity of PGRN. Our results support the use of serine protease inhibitors as antiinflammatory agents.

Neutrophil serine proteases (NSPs) in cytoplasmic granules of neutrophils are regarded as important antimicrobial defense weapons after engulfment and exposure of pathogens to the content of primary granules. Despite intensive studies on neutrophils during the last three decades, only three active serine proteases, neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (PR3) have been identified in these short-lived cells. Here, we report on the identification of a fourth serine protease (NSP4) with 39% identity to NE and PR3, but arginine specificity, yet sharing features like propeptide processing by dipeptidyl peptidase I, storage, and release as an active enzyme with the three active proteases. We established monoclonal antibodies against NSP4, excluded cross-reactivity to human granzymes, NE, CG, PR3, and azurocidin, and screened for NSP4 protein expression in various human tissues and blood leukocyte populations. Only granulocyte precursors and neutrophil populations from peripheral blood were positive. The content of NSP4 in neutrophil lysates, however, was about 20-fold lower compared with CG. Upon neutrophil activation, NSP4 was released into the supernatant. Profiling its specificity with peptide libraries fromEscherichia colirevealed a preference for arginine in P1; it cleaved Tyr-Arg-Phe-Arg-AMC and Ala-Pro-Nva-thiobenzyl esters. NSP4 was inhibited by α₁-proteinase inhibitor (α₁–antitrypsin), C1 inhibitor, and most efficiently by antithrombin-heparin, but not by elafin, secretory leukocyte protease inhibitor, α₁–antichymotrypsin, and monocyte-neutrophil elastase inhibitor. Functional specialization and preferred natural substrates of NSP4 remain to be determined to understand the biological interplay of all four NSPs during neutrophil responses.

Proteinase 3 (PR3), the target antigen of antineutrophil cytoplasm autoantibodies, which are found in patients with Wegener granulomatosis, is a neutrophil serine protease localized within cytoplasmic granules. Recently, the human neutrophil antigen NB1 was identified as a specific neutrophil cell surface receptor of PR3. We hypothesized that the unique hydrophobic cluster of PR3 that is not present on human neutrophil elastase and cathepsin G and presumably is also missing in other human PR3 homologs accounts for its binding to the NB1 receptor expressed on the cellular surface of NB1 cells. Instead of generating and testing various artificial human PR3 mutants, we cloned and expressed the very closely related gibbon (Hylobates pileatus) PR3 homolog, which did not bind to the human NB1 receptor. Moreover, a human-gibbon hybrid constructed from the N- and C-terminal half of the human and gibbon PR3, respectively, also did not interact with human NB1. The C-terminal half of gibbon PR3 differs only by 9 residues from human PR3, among which four closely spaced hydrophobic residues are substituted in a nonconservative manner (F166L, W218R, G219A, and L223H). The NB1-bound PR3 was active and was cleared from the surface by α-1-protease inhibitor. Conformational distortion of the hydrophobic 217-225 loop by α-1-protease inhibitor most likely triggers rapid solubilization.

How granzymes gain entry into the cytosol of target cells during killer cell attack has been the subject of several studies in the past, but the effective delivery mechanism during target cell encounter has not been clarified. Here we show that granzyme B (GzmB) mutants lacking binding to negatively charged, essentially heparan-sulfate-containing membrane receptors are poorly endocytosed yet are delivered to the cytosol with efficacy similar to that of WT GzmB. In a cell-based system GzmB-deficient natural killer cells provided perforin (pfn) by natural polarized secretion and synergized with externally added GzmB. Whereas receptor (heparan sulfate)-dependent endocytosis was dispensable, delivery of larger cargo like that of GzmB fusion proteins and GzmB-antibody complexes was restricted by their size. Our data support the model in which granzymes are primarily translocated through repairable membrane pores of finite size and not by the disruption of endocytosed vesicles. We conclude that structurally related translocators, i.e., perforin and cholesterol-dependent cytolysins, deliver deathly cargo across host cell membranes in a similar manner.