Grant R. Stenton

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 FcγR and FcεR, 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 β2 integrins, α4 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.

Role of intestinal mast cells in modulating gastrointestinal pathophysiology

OBJECTIVE This article reviews the current understanding of the pathophysiologic role of intestinal mast cells. DATA SOURCE Up to date English language publications on mast cell characteristics, heterogeneity and functions were used. Recent articles were used to develop and extend novel concepts about the role of intestinal mast cells. STUDY SELECTION Reference sources were selected because of their pertinence to the pathophysiological effects of mast cells in intestinal hypersensitivity. Recent publications on the following topics were emphasized: mast cell proteases in intestinal anaphylaxis; effects of nitric oxide in gastrointestinal pathophysiology; involvement of cytokines derived from mast cells in tissue damage and repair. RESULTS Mast cells are clearly implicated in the pathology of intestinal disease. Growing evidence suggests physiological roles for mast cells in the protection of tissues from inflammatory damage, and in intestinal maturation. Mast cells can release cytokines, such as tumour necrosis factor-alpha and interleukin-10, which were originally thought to contribute to inflammatory damage, but which may also have anti-inflammatory properties. Interestingly, mast cell function can be regulated by nitric oxide, and mast cells themselves are sources of this important mediator. Nitric oxide has protective as well as detrimental effects in the intestine. CONCLUSIONS Intestinal mast cells have physiologic regulatory effects in addition to their pathologic effects. However, relatively little is known about the mechanisms of these regulatory effects. Mast cells are likely in an ongoing fluctuating balance between physiological functions and pathological effects in normal individuals. Poorly known factors can create an imbalance and lead to pathologic reactions.

Aerosolized Syk Antisense Suppresses Syk Expression, Mediator Release from Macrophages, and Pulmonary Inflammation

Syk protein tyrosine kinase (PTK) is involved in signaling in leukocytes. In macrophages, Fcγ-receptor cross-linking induces Syk PTK phosphorylation and activation, resulting in Syk-dependent events required for phagocytosis and mediator release. We hypothesized that Syk antisense oligodeoxynucleotides (ASO) delivered by aerosol to rat lungs in vivo would depress Syk PTK expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation. RT-PCR and RT-in situ PCR demonstrated that aerosolized Syk ASO administration reduced Syk mRNA expression from alveolar macrophages compared with cells isolated from sham-treated rats. Western blot analysis confirmed that Syk PTK expression was reduced after Syk ASO treatment. Compared with sham-treated rats (scrambled oligodeoxynucleotide), Syk ASO treatment suppressed Fcγ-receptor-mediated nitric oxide (86.0 ± 8.3%) and TNF (73.1 ± 3.1%) production by alveolar macrophages stimulated with IgG-anti-IgG complexes. In contrast, Fcγ-receptor-induced IL-1β release was unaffected by Syk ASO treatment. Additionally, Syk ASO suppressed Ag-induced pulmonary inflammation, suggesting that Syk ASO may prove useful as an anti-inflammatory therapy in disorders such as asthma.

Frontline: Inhibition of allergen-induced pulmonary inflammation by the tripeptide feG: a mimetic of a neuro-endocrine pathway.

Interactions between the neuro‐endocrine system and immune system help maintain health. One interaction involves the superior cervical ganglia (SCG), which regulate the prohormone submandibular rat 1 (SMR1) produced by the submandibular gland (SMG). A peptide derived from SMR1, feG, has anti‐inflammatory activity, and modification to D‐isomer feG enhances bioactivity. We tested feG as a therapeutic agent for airways inflammation, using rats sensitized by OVA or Nippostrongylus brasiliensis (Nb). Treatment with feG but not fdG down‐regulated OVA‐challenge‐induced increases in bronchoalveolar lavage (BAL)‐derived macrophages, eosinophils and PMN (neutrophils) by 44%, 69% and 67%, respectively, at 24 h. We found that feG also reduced ICAM‐1 on BAL‐derived macrophages and eosinophils by 27% and 65%, and L‐selectin on PMN by 55% following OVA challenge. Furthermore, feG but not fdG reduced the OVA‐induced TNF increase in BAL fluid. We showed that feG also down‐regulated both hyper‐responsiveness to methacholine (by 27%) and microgranulomata formation in the lung parenchyma. In Nb‐challenged rats, feG treatment inhibited ex vivo allergen‐induced contraction of tracheal smooth muscle by up to 73%. In conclusion, feG, which is a mimetic of a peptide derived from a rat salivary gland prohormone, has anti‐inflammatory properties in allergic airways inflammation in Brown‐Norway rats. The role of the SCG‐SMG neuro‐endocrine pathway in allergic asthma and other inflammatory diseases requires additional study.

Activation of Macrophage CD8: Pharmacological Studies of TNF and IL-1β Production

Previously, we demonstrated that rat macrophages express CD8 and that Ab to CD8 stimulates NO production. We confirm that CD8 is expressed by rat macrophages and extend understanding of its functional significance. Activation of CD8α (OX8 Ab) on alveolar macrophages stimulated mRNA expression for TNF and IL-1β and promoted TNF and IL-1β secretion. Similarly, OX8 Ab (CD8α) stimulated NR8383 cells to secrete TNF, IL-1β, and NO. Activation of CD8β (Ab 341) on alveolar macrophages increased mRNA expression for TNF and IL-1β and stimulated secretion of TNF, but not IL-1β. Interestingly, anti-CD8 Abs did not stimulate IFN-γ or PGE2 production, or phagocytosis by macrophages. OX8 (CD8α)-induced TNF and IL-1β production by macrophages was blocked by inhibitors of protein tyrosine kinase(s), PP1, and genistein, but not by phosphatidylinositol-3 kinase inhibitor, wortmannin. Moreover, OX8 stimulated protein tyrosine kinase activity in NR8383 cells. Further analysis of kinase dependence using antisense to Syk kinase demonstrated that TNF, but not IL-1β, stimulation by CD8α is Syk dependent. By contrast, protein kinase C inhibitor Ro 31-8220 had no effect on OX8-induced TNF production, whereas OX8-induced IL-1β production was blocked by Ro 31-8220. Thus, there are distinct signaling mechanisms involved in CD8α (OX8)-induced TNF and IL-1β production. In summary, macrophages express CD8 molecules that, when activated, stimulate TNF and IL-1β expression, probably through mechanisms that include activation of Src and Syk kinases and protein kinase C. These findings identify a previously unknown pathway of macrophage activation likely to be involved in host defense and inflammation.

Involvement of Syk protein tyrosine kinase in LPS-induced responses in macrophages.

Syk kinase is best known as a critical component of immunoreceptor signaling in leukocytes. Activation of Syk following cross-linking of Fcγ and Fcε receptors on macrophages, mast cells, and other cells induces various inflammatory events. We hypothesized that Syk is involved in inflammatory responses induced by the lipopolysaccharide (LPS). We studied the role of Syk using its inhibition by antisense oligonucleotides, or small interfering RNA. Our data demonstrated that in vivo inhibition of Syk caused down-regulation of LPS-induced responses in rat alveolar macrophages. In in vitro experiments, inhibition of Syk in rat peritoneal macrophages, as well as in human myelomonocyte cell line THP-1 also caused a decrease in LPS-induced cytokine release. Our data support the hypothesis that, in macrophages, Syk is involved in LPS-induced intracellular signaling pathways leading to the release of pro-inflammatory mediators. Understanding the role of Syk in LPS-induced signaling may help in developing new therapeutic tools for inflammatory disorders.

A protease activated receptor-2 (PAR-2) activating peptide, tc-LIGRLO-NH2, induces protease release from mast cells: role in TNF degradation.

BackgroundMast cell (MC)-derived serine proteases have been implicated in a variety of inflammatory processes. We have previously shown that rat peritoneal MC (PMC) express mRNA for protease activated receptor 2 (PAR-2), a G-coupled receptor activated by trypsin-like proteases. Recent evidence also suggests that MC-induced inflammation can be mediated through PAR. Therefore, we hypothesized that specific PAR-2 agonist peptides (PAR-2ap) induce protease release from PMC.ResultsWestern blot analysis of PMC supernatants revealed that a PAR-2ap, tc-LIGRLO (10 μM), stimulated the release of rat MC protease (RMCP)-1, RMCP-5 and carboxypeptidase-A. The release was evident by 20 min but further increased up to 8 h. To study the biological effects of protease release we tested supernatants from tc-LIGRLO, tc-OLRGIL (inactive control peptide) and antigen-activated PMC for proteolytic activity by seeding with TNF (150 pg/ml), incubating for 8 h at 37°C, and measuring TNF remaining in the supernatants. Supernatants from tc-LIGRLO-stimulated PMC degraded 44 % of seeded TNF (n = 5). Moreover, this TNF proteolysis was dependent on the concentration of tc-LIGRLO used to stimulate PMC, and was significantly inhibited (94 %) by soybean trypsin inhibitor. Antigen and tc-OLRGIL induced no significant release of such proteolytic activity.ConclusionsThese data indicate that a PAR-2ap induces the release of proteases from mast cells, which may degrade extracellular cytokines and other substrates thus modulating the inflammatory response.

Reverse transcriptase in situ polymerase chain reaction for gene expression in rat mast cells and macrophages

Direct reverse transcriptase in situ polymerase chain reaction (RT-in situ PCR) of selected mRNA expression in rat mast cells (MC) and alveolar macrophages (AM) was optimized. Rat peritoneal mast cells (PMC), rat cultured mast cells (RCMC), rat bronchoalveolar lavage cells (BALC) or rat cultured alveolar macrophages (NR8383) were studied for the detection of mRNA for beta-actin, TNF-alpha and/or CD8alpha. Each type of cell has unique optimal conditions for RT-in situ PCR. The following parameters were carefully evaluated for optimization: protease digestion, DNAse digestion, heparinase digestion, RT, PCR cycle number and signal development with chromagen. Heparinase digestion was required for PMC mRNA detection because they contain large amounts of heparin proteoglycan, which is a potent inhibitor of RT and Taq polymerase enzymes. Only a few PCR cycles were needed to produce a cytoplasmic signal for mRNA transcripts in RCMC, whereas other types of cells (PMC, BALC and NR8383) needed at least 20 cycles for mRNA detection. The mRNA signal in PMC was localized to the perinuclear region, whereas mRNA in other cell types (RCMC, BALC and NR8383) were detected throughout the cytoplasm. Furthermore, modified Southern blot analysis for TNF-alpha in RCMC treated with RT-in situ PCR demonstrated the specificity of amplification product. The modified and optimized protocols for this procedure were successfully applied to detect and localize several mRNA transcripts in rat MC and AM. The approach is valuable and can be used to further study selected gene expression in these and other cell types.