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Mark Gilchrist

Publications and source records attributed to Mark Gilchrist.

5 recordsLinked to original sources
PubMed2003

Tetrahydrobiopterin, a critical factor in the production and role of nitric oxide in mast cells.

Mast cells (MC) are biologically potent, ubiquitously distributed immune cells with fundamental roles in host integrity and disease. MC diversity and function is regulated by exogenous nitric oxide; however, the production and function of endogenously produced NO in MC is enigmatic. We used rat peritoneal MC (PMC) as an in vivo model to examine intracellular NO production. Live cell confocal analysis of PMC using the NO-sensitive probe diaminofluorescein showed distinct patterns of intracellular NO formation with either antigen (Ag)/IgE (short term) or interferon-gamma (IFN-gamma) (long term). Ag/IgE-induced NO production is preceded by increased intracellular Ca2+, implying constitutive nitric-oxide synthase (NOS) activity. NO formation inhibits MC degranulation. NOS has obligate requirements for tetrahydrobiopterin (BH4), a product of GTP-cyclohydrolase I (CHI), IFN-gamma-stimulated PMC increased CHI mRNA, protein, and enzymatic activity, while decreasing CHI feedback regulatory protein mRNA, causing sustained NO production. Treatment with the CHI inhibitor, 2,4-diamino-6-hydroxypyrimidine, inhibited NO in both IFN-gamma and Ag/IgE systems, increasing MC degranulation. Reconstitution with the exogenous BH4 substrate, sepiapterin, restored NO formation and inhibited exocytosis. Thus, Ag/IgE and IFN-gamma induced intracellular NO plays a key role in MC mediator release, and alterations in NOS activity via BH4 availability may be critical to the heterogeneous responsiveness of MC.

Animals↗
PubMed2002

Inhibition of allergic inflammation in the airways using aerosolized antisense to Syk kinase.

Activation of the protein tyrosine kinase Syk is an early event that follows cross-linking of Fc gamma R and Fc epsilon R, leading to the release of biologically active molecules in inflammation. We reported previously that aerosolized Syk antisense oligodeoxynucleotides (ASO) depresses Syk expression in inflammatory cells, the release of mediators from alveolar macrophages, and pulmonary inflammation. To study the effect of Syk ASO in allergic inflammation and airway hyperresponsiveness, we used the Brown Norway rat model of OVA-induced allergic asthma. Syk ASO, delivered in a liposome, carrier/lipid complex by aerosol to rats, significantly inhibited the Ag-induced inflammatory cell infiltrate in the bronchoalveolar space, decreasing both neutrophilia and eosinophilia. The number of eosinophils in the lung parenchyma was also diminished. Syk ASO also depressed up-regulation of the expression of beta(2) integrins, alpha(4) integrin, and ICAM-1 in bronchoalveolar lavage leukocytes and reversed the Ag-induced decrease in CD62L expression on neutrophils. Furthermore, the increase in TNF levels in bronchoalveolar lavage following Ag challenge was significantly inhibited. Syk ASO also suppressed Ag-mediated contraction of the trachea in a complementary model. Thus, aerosolized Syk ASO suppresses many of the central components of allergic asthma and inflammation and may provide a new therapeutic approach.

Administration, Inhalation↗
PubMed2002

Proteinase-activated receptor (PAR)-1 and -2 agonists induce mediator release from mast cells by pathways distinct from PAR-1 and PAR-2.

Because thrombin-induced inflammation is partially mast cell-dependent and involves proteinase-activated receptors (PARs), we hypothesized that mast cells express PAR and can be stimulated with PAR-activating peptides (PAR-AP). We demonstrated that rat peritoneal mast cells expressed PAR-1 and PAR-2 mRNA, and that PAR-2AP (tc-LIGRLO-NH(2), 1 microm) induced 64.2 +/- 4.4% specific beta-hexosaminidase release from peritoneal mast cells, whereas another PAR-2AP (SLIGRL-NH(2), 10 microM), trypsin (40 U/ml), and mast cell tryptase (1.5 microg/ml) did not. PAR-1AP (ApfFRChaCitY-NH(2), 10 microM) (Cit) induced 11.7 +/- 3.7% specific beta-hexosaminidase release, whereas another PAR-1AP (TFLLR-NH(2), 40 microM) and human thrombin (10 U/ml) did not. PAR-AP, tc-LIGRLO-NH(2), and Cit increased the free intracellular Ca(2+) concentration, whereas trypsin, tryptase, thrombin, and other PAR-APs did not. Desensitization of Ca(2+) flux with different agonists suggests that although tc-LIGRLO-NH(2), Cit, and compound 48/80 have similar mechanisms of action, tc-LIGRLO-NH(2) also activates mast cells by a mechanism distinct from that of 48/80. Using benzalkonium chloride, which antagonizes the actions of 48/80 by competing for the same G(i) protein, we determined that benzalkonium chloride suppressed tc-LIGRLO-NH(2)-mediated (0.1 microM) beta-hexosaminidase release by 62%. Moreover, removal of sialic acid from peritoneal mast cells, using neuraminidase (2 U/ml), inhibited Cit- (10 microM, 52%) and tc-LIGRLO-NH(2) (0.5 microM, 29%)-mediated beta-hexosaminidase release. Thus, tc-LIGRLO-NH(2) and Cit have at least partially similar mechanisms of action as 48/80. PAR-AP may therefore activate mast cells via multiple mechanisms that are distinct from those of classical PAR-1 and PAR-2. The responsiveness of mast cells to PAR-AP via a non-PAR-1/non-PAR-2 mechanism complicates the interpretation of in vivo studies using these peptides.

Animals↗
PubMed2002

Nitric oxide synthase and nitric oxide production in in vivo-derived mast cells.

Nitric oxide (NO) is a potent mediator synthesized by a variety of cells involved in inflammatory reactions. We investigated the expression of NO synthase (NOS) in rat peritoneal mast cells (PMC). Small amounts of eNOS mRNA were detected basally, whereas neither mRNA for iNOS nor nNOS was detected in unstimulated PMC. Following stimulation by antigen, interferon-gamma (IFN-gamma), or anti-CD8 antibody, PMC up-regulated iNOS mRNA expression. In situ RT-PCR confirmed that iNOS mRNA originated from PMC. Production of iNOS protein was confirmed in stimulated PMC by immunohistochemistry. Upon stimulation with antigen, IFN-gamma, or anti-CD8, nitrite production was increased significantly (8.4+/-0.6, 7.6+/-0.9, and 6.6+/-0.9 microM/2x10(5) cells/48 h NO2-, respectively; P<0.01), whereas unstimulated PMC released 2.1 +/- 0.3 microM/2 x 10(5) cells/48 h NO2-. These findings demonstrate that in vivo-derived PMC transcribe and translate mRNA for NOS and produce NO.

Animals↗