Richard L. Stevens

Objective: Increased numbers of mast cells (MCs) that express β tryptases bound to heparin have been detected in the synovium of patients with rheumatoid arthritis (RA). The corresponding tryptases in mice are mouse MC protease 6 (mMCP‐6) and mMCP‐7. Although MCs have been implicated in RA and some animal models of arthritis, no direct evidence for a MC‐restricted tryptase in the pathogenesis of inflammatory arthritis has been shown. We created transgenic mice that lack heparin and different combinations of mMCP‐6 and mMCP‐7, to evaluate the roles of MC‐restricted tryptase–heparin complexes in an experimental model of arthritis.Methods: The methylated bovine serum albumin/interleukin‐1β (mBSA/IL‐1β) experimental protocol was used to induce inflammatory monarthritis in different mouse strains. Mice were killed at the time of peak disease on day 7, and histochemical methods were used to assess joint pathology.Results: Arthritis was induced in the knee joints of mBSA/IL‐1β–treated mMCP‐6⁺/mMCP‐7⁻ and mMCP‐6⁻/mMCP‐7⁺ C57BL/6 mice, and numerous activated MCs that had exocytosed the contents of their secretory granules were observed in the diseased mice. In contrast, arthritis was markedly reduced in heparin‐deficient mice and in mMCP‐6⁻/mMCP‐7⁻ C57BL/6 mice.Conclusion: MC‐derived tryptase–heparin complexes play important roles in mBSA/IL‐1β–induced arthritis. Because mMCP‐6 and mMCP‐7 can compensate for each other in this disease model, the elimination of both tryptases is necessary to reveal the prominent roles of these serine proteases in joint inflammation and destruction. Our data suggest that the inhibition of MC‐restricted tryptases could have therapeutic potential in the treatment of RA.

Summary:  Approximately 50% of the weight of a mature mast cell (MC) consists of varied neutral proteases stored in the cell’s secretory granules ionically bound to serglycin proteoglycans that contain heparin and/or chondroitin sulfate E/diB chains. Mouse MCs express the exopeptidase carboxypeptidase A3 and at least 15 serine proteases [designated as mouse MC protease (mMCP) 1–11, transmembrane tryptase/tryptase γ/protease serine member S (Prss) 31, cathepsin G, granzyme B, and neuropsin/Prss19]. mMCP‐6, mMCP‐7, mMCP‐11/Prss34, and Prss31 are the four members of the chromosome 17A3.3 family of tryptases that are preferentially expressed in MCs. One of the challenges ahead is to understand why MCs express so many different protease–proteoglycan macromolecular complexes. MC‐like cells that contain tryptase–heparin complexes in their secretory granules have been identified in the Ciona intestinalis and Styela plicata urochordates that appeared approximately 500 million years ago. Because sea squirts lack B cells and T cells, it is likely that MCs and their tryptase–proteoglycan granule mediators initially appeared in lower organisms as part of their innate immune system. The conservation of MCs throughout evolution suggests that some of these protease–proteoglycan complexes are essential to our survival. In support of this conclusion, no human has been identified that lacks MCs. Moreover, transgenic mice lacking the β‐tryptase mMCP‐6 are unable to combat a Klebsiella pneumoniae infection effectively. Here we summarize the nature and function of some of the tryptase–serglycin proteoglycan complexes found in mouse and human MCs.

Tryptase ϵ is a member of the chromosome 16p13.3 family of human serine proteases that is preferentially expressed by epithelial cells. Recombinant pro–tryptase ϵ was generated to understand how the exocytosed zymogen might be activated outside of the epithelial cell, as well as to address its possible role in normal and diseased states. Using expression/site-directed mutagenesis approaches, we now show that Lys²⁰, Cys⁹⁰, and Asp⁹² in the protease9s substrate-binding cleft regulate its enzymatic activity. We also show that Arg^-1 in the propeptide domain controls its ability to autoactivate. In vitro studies revealed that recombinant tryptase ϵ possesses a restricted substrate specificity. Once activated, tryptase ϵ cannot be inhibited effectively by the diverse array of protease inhibitors present in normal human plasma. Moreover, this epithelium protease is not highly susceptible to α1-antitrypsin or secretory leukocyte protease inhibitor, which are present in the lung. Recombinant tryptase ϵ could not cleave fibronectin, vitronectin, laminin, single-chain tissue-type plasminogen activator, plasminogen, or any prominent serum protein. Nevertheless, tryptase ϵ readily converted single-chain pro–urokinase-type plasminogen activator (pro-uPA/scuPA) into its mature, enzymatically active protease. Tryptase ϵ also was able to induce pro-uPA–expressing smooth muscle cells to increase their migration through a basement membrane–like extracellular matrix. The ability to activate uPA in the presence of varied protease inhibitors suggests that tryptase ϵ plays a prominent role in fibrinolysis and other uPA-dependent reactions in the lung.