Jennifer A. Cairns

Mast cell tryptase stimulates the synthesis of type I collagen in human lung fibroblasts.

Mast cell activation is a characteristic feature of chronic inflammation, a condition that may lead to fibrosis as a result of increased collagen synthesis by fibroblasts. We have investigated the potential of tryptase, the major protease of human mast cells, to stimulate collagen synthesis in the human lung fibroblast cell line MRC-5. Tryptase was isolated from human lung tissue by ion-exchange and affinity chromatography. At concentrations of 18 and 36 mU/ml, tryptase stimulated both an increase in cell numbers, and a fivefold increase in DNA synthesis as determined by methyl-[3H]thymidine incorporation. Similar concentrations of tryptase resulted in a 2.5-fold increase in collagen synthesis as determined both by incorporation of [3H]proline into collagen, and by assay of hydroxyproline concentrations in the supernatants. There was also a twofold increase in collagenolytic activity in the culture medium after tryptase treatment, indicating that the increase in collagen synthesis was not a consequence of decreased collagenase production. All of these actions of tryptase were reduced in the presence of the protease inhibitors leupeptin and benzamidine hydrochloride, indicating a requirement for an active catalytic site. SDS-PAGE and autoradiographic analysis of the [3H]collagen produced by the cells revealed it to be predominantly type I collagen. Our findings suggest that the release of tryptase from activated mast cells may provide a signal for abnormal fibrosis in inflammatory disease.

A Polymorphic Protease-activated Receptor 2 (PAR2) Displaying Reduced Sensitivity to Trypsin and Differential Responses to PAR Agonists

Protease-activated receptor 2 (PAR2) is a trypsin-activated member of a family of G-protein-coupled PARs. We have identified a polymorphic form of human PAR2 (PAR2F240S) characterized by a phenylalanine to serine mutation at residue 240 within extracellular loop 2, with allelic frequencies of 0.916 (Phe240) and 0.084 (Ser240) for the wild-type and mutant alleles, respectively. Elevations in intracellular calcium were measured in permanently transfected cell lines expressing the receptors. PAR2F240S displayed a significant reduction in sensitivity toward trypsin (∼3.7-fold) and the PAR2-activating peptides, SLIGKV-NH2 (∼2.5-fold) and SLIGRL-NH2(∼2.8-fold), but an increased sensitivity toward the selective PAR2 agonist,trans-cinnamoyl-LIGRLO-NH2(∼4-fold). Increased sensitivity was also observed toward the selective PAR-1 agonist, TFLLR-NH2 (∼7-fold), but not to other PAR-1 agonists tested. Furthermore, we found that TLIGRL-NH2 and a PAR4-derived peptide,trans-cinnamoyl-YPGKF-NH2, were selective PAR2F240S agonists. By introducing the F240S mutation into rat PAR2, we observed shifts in agonist potencies that mirrored the human PAR2F240S, suggesting that Phe240 is involved in determining agonist specificity of PAR2. Finally, differences in receptor signaling were paralleled in a cell growth assay. We suggest that the distinct pharmacological profile induced by this polymorphism will have important implications for the design of PAR-targeted agonists/antagonists and may contribute to, or be predictive of, an inflammatory disease.

Human mast cell tryptase stimulates the release of an IL-8-dependent neutrophil chemotactic activity from human umbilical vein endothelial cells (HUVEC)

Tryptase, the major product of human mast cell activation, is a potent stimulus of vascular leakage and neutrophil accumulation in vivo in animal studies, but the mechanisms of action remain unclear. Using HUVEC cultures we have sought to investigate the potential of tryptase to alter monolayer permeability or induce the release of neutrophil chemotactic activity. Tryptase (1–100 mU/ml) failed to alter the permeability of endothelial cell monolayers as assessed by albumin flux over 1 h. However, supernatants from endothelial cells treated with tryptase (1–50 mU/ml) for a 24‐h period induced neutrophil migration across Transwell filters, with maximal migration observed at 10 mU/ml tryptase. Pretreatment of tryptase with the protease inhibitor leupeptin abolished the chemotactic activity, indicating a dependence on the catalytic site. Moreover, this effect was abolished by addition of an IL‐8 neutralizing antibody, suggesting that IL‐8 release makes an important contribution to the chemotactic activity. The interaction of mast cell tryptase with endothelial cells could be important in stimulating the ingress of neutrophils following mast cell activation in inflammatory disease.

Interaction of Human Mast Cell Tryptase with Endothelial Cells to Stimulate Inflammatory Cell Recruitment

125I-albumin flux. The structurally related serine protease, thrombin, increased permeability, though tryptase either in the presence or absence of heparin and with incubation periods up to 1 h was without effect. These observations are consistent with the idea that tryptase-induced microvascular leakage is not mediated by a direct effect of this protease on vascular endothelial cells. The finding that the size of the reactions induced in guinea pig skin may be reduced by pretreating the animals with antihistamines [2] has prompted the suggestion that the increase in microvascular permeability may be a consequence of mast cell degranulation, and tryptase has been demonstrated to stimulate histamine release from both guinea pig and human mast cells [2, 6]. Tryptase was found by RT-PCR to increase expression of mRNA for IL-8 and for IL-1 â though that for GM-CSF, IL-6, IL-15 and RANTES was unaffected. There was a dose-dependent release into the supernatants of IL-8, which was maximal at 24 h with a tryptase concentration of 50 mU/ml. The effect was dependent on an intact catalytic site, being reduced significantly by preincubation of tryptase with the protease inhibitors leupeptin or benzamidine, heat inactivating the enzyme, or depleting samples of tryptase by immunoabsorption with a tryptase-specific antibody AA5. No detectable increases in IL-1â protein (precursor or mature) were detected in the supernatants or lysates of tryptase-treated HUVEC. Incubation of HUVEC with tryptase stimulated the release of neutrophil chemotactic activity in studies of 51Crlabelled neutrophil transmigration through naked Transwell filters. Neutrophil transmigration following incubation of Mast Cell Biology

Alternate mRNA Splicing in Multiple Human Tryptase Genes Is Predicted to Regulate Tetramer Formation

Tryptases are serine proteases that are thought to be uniquely and proteolytically active as tetramers. Crystallographic studies reveal that the active tetramer is a flat ring structure composed of four monomers, with their active sites arranged around a narrow central pore. This model explains why many of the preferred substrates of tryptase are short peptides; however, it does not explain how tryptase cleaves large protein substrates such as fibronectin, although a number of studies have reported in vitro mechanisms for generating active monomers that could digest larger substrates. Here we suggest that alternate mRNA splicing of human tryptase genes generates active tryptase monomers (or dimers). We have identified a conserved pattern of alternate splicing in four tryptase alleles (αII, βI, βIII, and δI), representing three distinct tryptase gene loci. When compared with their full-length counterparts, the splice variants use an alternate acceptor site within exon 4. This results in the deletion of 27 nucleotides within the central coding sequence and 9 amino acids from the translated protein product. Although modeling suggests that the deletion can be easily accommodated by the enzymes structurally, it is predicted to alter the specificity by enlarging the S1′ or S2′ binding pocket and results in the complete loss of the “47 loop,” reported to be critical for the formation of tetramers. Although active monomers can be generated in vitro using a range of artificial conditions, we suggest that alternate splicing is the in vivo mechanism used to generate active tryptase that can cleave large protein substrates.

Mast cell and eosinophil proteases in the chronic allergic inflammatory response

Publisher Summary The cleavage of peptides and proteins by proteases is crucial for the maintenance of normal body function, but uncontrolled proteolysis in the tissues can have serious consequences. An imbalance between certain proteases and their inhibitors has long been implicated in the pathogenesis of emphysema as well as in certain other chronic conditions. An increased degree of mast cell activation and increased numbers of eosinophils, frequently in an activated state, have become regarded as some of the defining characteristics of allergic disease. Measurements of tryptase, a serine protease unique to mast cells, histamine and prostaglandin D2 in biological fluids have helped to establish the role of mast cells in such conditions. Thus, tryptase levels are increased in bronchoalveolar lavage (BAL) fluid collected from patients with atopic or intrinsic asthma as well as with certain other respiratory conditions, indicating persistent degranulation of pulmonary mast cells. An increase in BAL fluid concentrations of tryptase has been detected within minutes of the introduction of allergen into the lower airways.