Neutrophilia, gelatinase release and microvascular leakage induced by human mast cell tryptase in a mouse model: Lack of a role of protease-activated receptor 2 (PAR2).

BACKGROUND: Tryptase, the most abundant protease of the human mast cell, has been implicated as a key mediator of allergic inflammation that acts through activation of PAR2. OBJECTIVES: To investigate the contribution of PAR2 in the pro-inflammatory actions mediated by tryptase in a mice model. METHODS: We have injected recombinant human ßII-tryptase into the peritoneum of PAR2-deficient and wild-type C57BL/6 mice. After 6, 12 and 24 hours, mice were killed, peritoneal lavage performed and inflammatory changes investigated. RESULTS: Tryptase stimulated an increase in neutrophil numbers in the peritoneum, but responses did not differ between PAR2-deficient and wild-type mice. Heat inactivation of tryptase or pre-incubation with a selective tryptase inhibitor reduced neutrophilia, but neutrophil accumulation was not elicited with a peptide agonist of PAR2 (SLIGRL-NH2 ). Zymography indicated that tryptase stimulated the release of matrix metalloproteinases (MMP) 2 and 9 in the peritoneum of both mouse strains. Studies involving immunomagnetic isolation of neutrophils suggested that neutrophils represent the major cellular source of tryptase-induced MMP2 and MMP9. At 24 hours after tryptase injection, there was increased microvascular leakage as indicated by high levels of albumin in peritoneal lavage fluid, and this appeared to be partially abolished by heat-inactivating tryptase or addition of a protease inhibitor. There was no corresponding increase in levels of histamine or total protein. The extent of tryptase-induced microvascular leakage or gelatinase release into the peritoneum did not differ between PAR2-deficient and wild-type mice. CONCLUSIONS: Our findings indicate that tryptase is a potent stimulus for neutrophil accumulation, MMP release and microvascular leakage. Although these actions required an intact catalytic site, the primary mechanism of tryptase in vivo would appear to involve processes independent of PAR2.

Alpha-tryptase gene variation is associated with levels of circulating IgE and lung function in asthma.

BACKGROUND: Tryptase, a major secretory product of human mast cells has been implicated as a key mediator of allergic inflammation. Genetic variation in the tryptases is extensive, and α-tryptase, an allelic variant of the more extensively studied ß-tryptase, is absent in substantial numbers of the general population. The degree to which α-tryptase expression may be associated with asthma has not been studied. We have investigated the α-tryptase gene copy number variation and its potential associations with phenotypes of asthma. OBJECTIVES: Caucasian families (n = 341) with at least two asthmatic siblings (n = 1350) were genotyped for the α-tryptase alleles, using high-resolution melting assays. Standards for the possible α-/ß-tryptase ratios were constructed by cloning α-and ß-tryptase PCR products to generate artificial templates. Association analysis of asthma affection status and related phenotypes [total and allergen-specific serum IgE, bronchial hyperresponsiveness to methacholine, forced expiratory volume in 1s (FEV1 ) and atopy and asthma severity scores] was undertaken using family-based association tests (FBAT). RESULTS: Four consistent melting patterns for the α-tryptase genotype were identified with alleles carrying null, one or two copies of the α-tryptase allele. Possessing one copy of α-tryptase was significantly associated with lower serum levels of total and dust mite-specific IgE levels and higher FEV1 measurements, while two copies were related to higher serum concentrations of total and dust mite-specific IgE and greater atopy severity scores. CONCLUSIONS AND CLINICAL RELEVANCE: Associations of α-tryptase copy number with serum IgE levels, atopy scores and bronchial function may reflect roles for tryptases in regulating IgE production and other processes in asthma.

Vasoactive intestinal peptide induces proliferation of human hepatocytes.

OBJECTIVES: Proliferation of hepatocytes in vitro can be stimulated by growth factors such as epidermal growth factor (EGF), but the role of vasoactive intestinal peptide (VIP) remains unclear. We have investigated the effect of VIP on maintenance and proliferation of human hepatocytes. MATERIALS AND METHODS: Human hepatocytes were isolated from liver specimens obtained from patients undergoing liver surgery. Treatment with VIP or EGF was started 24 h after plating and continued for 3 or 5 d. DNA replication was investigated by Bromodeoxyuridine (BrdU) incorporation and cell viability detected by MTT assay. Cell lysate was analysed by western blotting and RT-PCR. Urea and albumin secretion into the culture supernatants were measured. RESULTS: VIP increased DNA replication in hepatocytes in a dose-dependant manner, with a peak response at day 3 of treatment. VIP treatment was associated with an increase in mRNA expression of antigen identified by monoclonal antibody Ki-67 (MKI-67) and Histone Cluster 3 (H3) genes. Western blotting analysis showed that VIP can induce a PKA/B-Raf dependant phosphorylation of extracellular signal-regulated kinases (ERK). Although EGF can maintain hepatocyte functions up to day 5, no marked efffect was found with VIP. CONCLUSIONS: VIP induces proliferation of human hepatocytes with little or no effect on hepatocyte differentiation. Further investigation of the role of VIP is required to determine if it may ultimately support therapeutic approaches of liver disease.